Adeno-associated virus (aav) serotype 8 sequences, vectors containing same, and uses therefor

ABSTRACT

Sequences of a serotype 8 adeno-associated virus and vectors and host cells containing these sequences are provided. Also described are methods of using such host cells and vectors in production of rAAV particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/084,615, filed Mar. 30, 2016, which is a divisional of U.S. patent application Ser. No. 14/598,462, filed Jan. 16, 2015, now U.S. Pat. No. 9,493,788, issued Nov. 15, 2016, which is a divisional of U.S. patent application Ser. No. 11/981,022, filed Oct. 31, 2007, now U.S. Pat. No. 8,962,330, issued Feb. 24, 2015, which is a continuation of U.S. patent application Ser. No. 11/899,500, filed Sep. 6, 2007, now U.S. Pat. No. 7,790,449, issued Sep. 7, 2010, which is a continuation of U.S. patent application Ser. No. 10/423,704, filed Apr. 25, 2003, now U.S. Pat. No. 7,282,199, issued Oct. 16, 2007, which is a continuation-in-part of International Patent Application No. PCT/US02/33630, filed Nov. 12, 2002, which claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 60/386,122, filed Jun. 5, 2002, U.S. Provisional Patent Application No. 60/377,133, filed May 1, 2002, and U.S. Provisional Patent Application No. 60/341,151, filed Dec. 17, 2001, which applications are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. P30 DK 47757-09 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases, and Grant No. P01 HL 59407-03 awarded by the National Heart, Lung, and Blood Institute. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

The Sequence Listing material filed in electronic form herewith is hereby incorporated by reference. This file is labeled “UPN_O2733C4D2C1_ST25.txt”, was created on Apr. 21, 2017, and is 50,047 bytes (48.8 KB).

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV), a member of the Parvovirus family, is a small nonenveloped, icosahedral virus with single-stranded linear DNA genomes of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus, Dependovirus, because the virus was discovered as a contaminant in purified adenovirus stocks. AAV's life cycle includes a latent phase at which AAV genomes, after infection, are site specifically integrated into host chromosomes and an infectious phase in which, following either adenovirus or herpes simplex virus infection, the integrated genomes are subsequently rescued, replicated, and packaged into infectious viruses. The properties of non-pathogenicity, broad host range of infectivity, including non-dividing cells, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer.

Recent studies suggest that AAV vectors may be the preferred vehicle for gene delivery. To date, there have been 6 different serotypes of AAVs isolated from human or non-human primates (NHP) and well characterized. Among them, human serotype 2 is the first AAV that was developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in different target tissues and animal models. Clinical trials of the experimental application of AAV2 based vectors to some human disease models are in progress, and include such diseases as cystic fibrosis and hemophilia B.

What are desirable are AAV-based constructs for gene delivery.

SUMMARY OF THE INVENTION

In one aspect, the invention provides novel AAV sequences, compositions containing these sequences, and uses therefor. Advantageously, these compositions are particularly well suited for use in compositions requiring re-administration of rAAV for therapeutic or prophylactic purposes.

These and other aspects of the invention will be readily apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1C are the nucleic acid sequences of the rep and cap regions of AAV8 [SEQ ID NO:1].

FIGS. 2A through 2C are the amino acid sequences of the AAV8 capsid vp1 protein [SEQ ID NO:2], provided in alignment with the vp1 of the published sequences of AAV2 [SEQ ID NO:4], AAV1 [SEQ ID NO:5], and AAV3 [SEQ ID NO:6], and newly identified AAV serotypes AAV7 [SEQ ID NO: 8] and AAV9 [SEQ ID NO:7]. The alignment was performed using the Clustal W program, with the number of AAV2 used for reference. Underlining and bold at the bottom sequence of the alignment indicates cassettes of identity. The dots in the alignment indicate that the amino acids are missing at the positions in the alignment as compared to AAV2 VP1.

FIGS. 3A through 3C are the amino acid sequences of the AAV8 rep proteins [SEQ ID NO:3].

DETAILED DESCRIPTION OF THE INVENTION

The invention provides the nucleic acid sequences and amino acids of a novel AAV serotype, AAV8. Also provided are fragments of these AAV sequences. Each of these fragments may be readily utilized in a variety of vector systems and host cells. Among desirable AAV8 fragments are the cap proteins, including the vp1, vp2, vp3 and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells. Such fragments may be used alone, in combination with other AAV8 sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. In one particularly desirable embodiment, a vector contains the AAV8 cap and/or rep sequences of the invention.

The AAV8 sequences and fragments thereof are useful in production of rAAV, and are also useful as antisense delivery vectors, gene therapy vectors, or vaccine vectors. The invention further provides nucleic acid molecules, gene delivery vectors, and host cells which contain the AAV8 sequences of the invention.

Suitable fragments can be determined using the information provided herein. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs, such as “Clustal W”, accessible through Web Servers on the internet. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art which can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta, a program in GCG Version 6.1. Fasta provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference. Similar programs are available for amino acid sequences, e.g., the “Clustal X” program. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs.

The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

The term “substantial homology” or “substantial similarity,” when referring to amino acids or fragments thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions with another amino acid (or its complementary strand), there is amino acid sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is over full-length sequence, or a protein thereof, e.g., a cap protein, a rep protein, or a fragment thereof which is at least 8 amino acids, or more desirably, at least 15 amino acids in length. Examples of suitable fragments are described herein.

By the term “highly conserved” is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art.

The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. Similarly, “percent sequence identity” may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof. Suitably, a fragment is at least about 8 amino acids in length, and may be up to about 700 amino acids. Examples of suitable fragments are described herein.

As described herein, the vectors of the invention containing the AAV capsid proteins of the invention are particularly well suited for use in applications in which the neutralizing antibodies diminish the effectiveness of other AAV serotype based vectors, as well as other viral vectors. The rAAV vectors of the invention are particularly advantageous in rAAV readministration and repeat gene therapy.

These and other embodiments and advantages of the invention are described in more detail below. As used throughout this specification and the claims, the terms “comprising” and “including” are inclusive of other components, elements, integers, steps and the like. Conversely, the term “consisting” and its variants are exclusive of other components, elements, integers, steps and the like.

I. AAV Serotype 8 Sequences

-   A. Nucleic Acid Sequences

The AAV8 nucleic acid sequences of the invention include the DNA sequences of FIG. 1 [SEQ ID NO: 1], which consists of 4393 nucleotides. The AAV8 nucleic acid sequences of the invention further encompass the strand which is complementary to FIG. 1 [SEQ ID NO: 1], as well as the RNA and cDNA sequences corresponding to FIG. 1 [SEQ ID NO: 1] and its complementary strand. Also included in the nucleic acid sequences of the invention are natural variants and engineered modifications of FIG. 1 [SEQ ID NO: 1] and its complementary strand. Such modifications include, for example, labels which are known in the art, methylation, and substitution of one or more of the naturally occurring nucleotides with a degenerate nucleotide.

Further included in this invention are nucleic acid sequences which are greater than about 90%, more preferably at least about 95%, and most preferably at least about 98 to 99% identical or homologous to FIG. 1 [SEQ ID NO:1].

Also included within the invention are fragments of FIG. 1 [SEQ ID NO: 1], its complementary strand, cDNA and RNA complementary thereto. Suitable fragments are at least 15 nucleotides in length, and encompass functional fragments, i.e., fragments which are of biological interest. Such fragments include the sequences encoding the three variable proteins (vp) of the AAV8 capsid which are alternative splice variants: vp1 [nt 2121 to 4337 of FIG. 1, SEQ ID NO:1]; vp2 [nt 2532 to 4337 of FIG. 1, SEQ ID NO:1]; and vp 3 [nt 2730 to 4337 of FIG. 1, SEQ ID NO:1]. Other suitable fragments of FIG. 1 [SEQ ID NO:1], include the fragment which contains the start codon for the AAV8 capsid protein, and the fragments encoding the hypervariable regions of the vp1 capsid protein, which are described herein.

Still other fragments include those encoding the rep proteins, including rep 78 [initiation codon located at nt 227 of FIG. 1, SEQ ID NO:1], rep 68 [initiation codon located at nt 227 of FIG. 1, SEQ ID NO:1], rep 52 [initiation codon located at nt 905 of FIG. 1, SEQ ID NO:1], and rep 40 [initiation codon located at nt 905 of FIG. 1, SEQ ID NO:1]. Other fragments of interest may include the AAV8 inverted terminal repeat which can be identified by the methods described herein, AAV P19 sequences, AAV8 P40 sequences, the rep binding site, and the terminal resolute site (TRS). Still other suitable fragments will be readily apparent to those of skill in the art.

In addition to including the nucleic acid sequences provided in the figures and Sequence Listing, the present invention includes nucleic acid molecules and sequences which are designed to express the amino acid sequences, proteins and peptides of the AAV serotypes of the invention. Thus, the invention includes nucleic acid sequences which encode the following novel AAV amino acid sequences and artificial AAV serotypes generated using these sequences and/or unique fragments thereof.

As used herein, artificial AAV serotypes include, without limitation, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a novel AAV sequence of the invention (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from another AAV serotype (known or novel), non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. An artificial AAV serotype may be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid.

-   B. AAV8 Amino Acid Sequences, Proteins and Peptides

The invention further provides proteins and fragments thereof which are encoded by the AAV8 nucleic acids of the invention, and AAV8 amino acids which are generated by other methods. The invention further encompasses AAV serotypes generated using sequences of the novel AAV serotype of the invention, which are generated using synthetic, recombinant or other techniques known to those of skill in the art. The invention is not limited to novel AAV amino acid sequences, peptides and proteins expressed from the novel AAV nucleic acid sequences of the invention and encompasses amino acid sequences, peptides and proteins generated by other methods known in the art, including, e.g., by chemical synthesis, by other synthetic techniques, or by other methods. For example, the sequences of any of be readily generated using a variety of techniques.

Suitable production techniques are well known to those of skill in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively, peptides can also be synthesized by the well known solid phase peptide synthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962); Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62). These and other suitable production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.

Particularly desirable proteins include the AAV capsid proteins, which are encoded by the nucleotide sequences identified above. The AAV capsid is composed of three proteins, vp1, vp2 and vp3, which are alternative splice variants. The full-length sequence provided in FIG. 2 is that of vp1. The AAV8 capsid proteins include vp1 [aa 1 to 738 of SEQ ID NO:2], vp2 [aa 138 to 738 of SEQ ID NO:2], and vp3 [aa 204 to 738 of SEQ ID NO: 2] and functional fragments thereof Other desirable fragments of the capsid protein include the constant and variable regions, located between hypervariable regions (HPV). Other desirable fragments of the capsid protein include the HPV themselves.

An algorithm developed to determine areas of sequence divergence in AAV2 has yielded 12 hypervariable regions (HVR) of which 5 overlap or are part of the four previously described variable regions. [Chiorini et al, J. Virol, 73:1309-19 (1999); Rutledge et al, J Virol., 72:309-319] Using this algorithm and/or the alignment techniques described herein, the HVR of the novel AAV serotypes are determined. For example, with respect to the number of the AAV2 vp1 [SEQ ID NO:4], the HVR are located as follows: HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa 262-264; HVR4, aa 381-383; HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa500-504; HVR8, aa 514-522; HVR9, aa 534-555; HVR10, aa 581-594; HVR11, aa 658-667; and HVR12, aa 705-719. Using the alignment provided herein performed using the Clustal X program at default settings, or using other commercially or publicly available alignment programs at default settings, one of skill in the art can readily determine corresponding fragments of the novel AAV capsids of the invention.

Still other desirable fragments of the AAV8 capsid protein include amino acids 1 to 184 of SEQ ID NO: 2, amino acids 199 to 259; amino acids 274 to 446; amino acids 603 to 659; amino acids 670 to 706; amino acids 724 to 736 of SEQ ID NO:2; aa 185-198; aa 260-273; aa447-477; aa495-602; aa660-669; and aa707-723. Additionally, examples of other suitable fragments of AAV capsids include, with respect to the numbering of AAV2 [SEQ ID NO:4], aa 24-42, aa 25-28; aa 81-85; aa133-165; aa 134-165; aa 137-143; aa 154-156; aa 194-208; aa 261-274; aa 262-274; aa 171-173; aa 413-417; aa 449-478; aa 494-525; aa 534-571; aa 581-601; aa 660-671; aa 709-723. Still other desirable fragments include, for example, in AAV7, amino acids 1 to 184 of SEQ ID NO:2, amino acids 199 to 259; amino acids 274 to 446; amino acids 603 to 659; amino acids 670 to 706; amino acids 724 to 736; aa 185 to 198; aa 260 to 273; aa447 to 477; aa495 to 602; aa660 to 669; and aa707 to 723. Using the alignment provided herein performed using the Clustal X program at default settings, or using other commercially or publicly available alignment programs at default settings, one of skill in the art can readily determine corresponding fragments of the novel AAV capsids of the invention.

Still other desirable AAV8 proteins include the rep proteins include rep68/78 and rep40/52 [located within aa 1 to 625 of SEQ ID NO: 3]. Suitable fragments of the rep proteins may include aa 1 to 102; aa 103 to 140; aa 141 to 173; aa 174 to 226; aa 227 to 275; aa 276 to 374; aa 375 to 383; aa 384 to 446; aa 447 to 542; aa 543 to 555;

aa 556 to 625, of SEQ ID NO: 3.

Suitably, fragments are at least 8 amino acids in length. However, fragments of other desired lengths may be readily utilized. Such fragments may be produced recombinantly or by other suitable means, e.g., chemical synthesis.

The invention further provides other AAV8 sequences which are identified using the sequence information provided herein. For example, given the AAV8 sequences provided herein, infectious AAV8 may be isolated using genome walking technology (Siebert et al., 1995, Nucleic Acid Research, 23:1087-1088, Friezner-Degen et al., 1986, J Biol. Chem. 261:6972-6985, BD Biosciences Clontech, Palo Alto, Calif.). Genome walking is particularly well suited for identifying and isolating the sequences adjacent to the novel sequences identified according to the method of the invention. This technique is also useful for isolating inverted terminal repeat (ITRs) of the novel AAV8 serotype, based upon the novel AAV capsid and rep sequences provided herein.

The sequences, proteins, and fragments of the invention may be produced by any suitable means, including recombinant production, chemical synthesis, or other synthetic means. Such production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.

IV. Production of rAAV with AAV8 Capsids

The invention encompasses novel, wild-type AAV8, the sequences of which are free of DNA and/or cellular material with these viruses are associated in nature. In another aspect, the present invention provides molecules which utilize the novel AAV sequences of the invention, including fragments thereof, for production of molecules useful in delivery of a heterologous gene or other nucleic acid sequences to a target cell.

In another aspect, the present invention provides molecules which utilize the AAV8 sequences of the invention, including fragments thereof, for production of viral vectors useful in delivery of a heterologous gene or other nucleic acid sequences to a target cell.

The molecules of the invention which contain AAV8 sequences include any genetic element (vector) which may be delivered to a host cell, e.g., naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in a non-viral delivery vehicle (e.g., a lipid-based carrier), virus, etc. which transfer the sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

In one embodiment, the vectors of the invention contain, at a minimum, sequences encoding an AAV8 capsid or a fragment thereof. In another embodiment, the vectors of the invention contain, at a minimum, sequences encoding an AAV8 rep protein or a fragment thereof Optionally, such vectors may contain both AAV cap and rep proteins. In vectors in which both AAV rep and cap are provides, the AAV rep and AAV cap sequences can both be of AAV8 origin. Alternatively, the present invention provides vectors in which the rep sequences are from an AAV serotype which differs from that which is providing the cap sequences. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form a chimeric AAV vector. Optionally, the vectors of the invention further contain a minigene comprising a selected transgene which is flanked by AAV 5′ ITR and AAV 3′ ITR.

Thus, in one embodiment, the vectors described herein contain nucleic acid sequences encoding an intact AAV capsid which may be from a single AAV serotype (e.g., AAV8). Such a capsid may comprise amino acids 1 to 738 of SEQ ID NO:2. Alternatively, these vectors contain sequences encoding artificial capsids which contain one or more fragments of the AAV8 capsid fused to heterologous AAV or non-AAV capsid proteins (or fragments thereof). These artificial capsid proteins are selected from non-contiguous portions of the AAV8 capsid or from capsids of other AAV serotypes. For example, a rAAV may have a capsid protein comprising one or more of the AAV8 capsid regions selected from the vp2 and/or vp3, or from vp 1, or fragments thereof selected from amino acids 1 to 184, amino acids 199 to 259; amino acids 274 to 446; amino acids 603 to 659; amino acids 670 to 706; amino acids 724 to 738 of the AAV8 capsid, SEQ ID NO: 2. In another example, it may be desirable to alter the start codon of the vp3 protein to GTG. Alternatively, the rAAV may contain one or more of the AAV serotype 8 capsid protein hypervariable regions which are identified herein, or other fragment including, without limitation, aa 185-198; aa 260-273; aa447-477; aa495-602; aa660-669; and aa707-723 of the AAV8 capsid. See, SEQ ID NO: 2. These modifications may be to increase expression, yield, and/or to improve purification in the selected expression systems, or for another desired purpose (e.g., to change tropism or alter neutralizing antibody epitopes).

The vectors described herein, e.g., a plasmid, are useful for a variety of purposes, but are particularly well suited for use in production of a rAAV containing a capsid comprising AAV sequences or a fragment thereof These vectors, including rAAV, their elements, construction, and uses are described in detail herein.

In one aspect, the invention provides a method of generating a recombinant adeno-associated virus (AAV) having an AAV serotype 8 capsid, or a portion thereof. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an adeno-associated virus (AAV) serotype 8 capsid protein, or fragment thereof, as defined herein; a functional rep gene; a minigene composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the minigene into the AAV8 capsid protein.

The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigene, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contains the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functions required for producing the rAAV of the invention may be delivered to the packaging host cell in the form of any genetic element which transfer the sequences carried thereon. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al, J Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

Unless otherwise specified, the AAV ITRs, and other selected AAV components described herein, may be readily selected from among any AAV serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9 and the novel serotype of the invention, AAV8. These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from an AAV serotype. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like.

-   -   A. The Minigene         -   The minigene is composed of, at a minimum, a transgene and             its regulatory sequences, and 5′ and 3′ AAV inverted             terminal repeats (ITRs). In one desirable embodiment, the             ITRs of AAV serotype 2 are used. However, ITRs from other             suitable serotypes may be selected. It is this minigene             which is packaged into a capsid protein and delivered to a             selected host cell.         -   1. The Transgene             -   The transgene is a nucleic acid sequence, heterologous                 to the vector sequences flanking the transgene, which                 encodes a polypeptide, protein, or other product, of                 interest. The nucleic acid coding sequence is                 operatively linked to regulatory components in a manner                 which permits transgene transcription, translation,                 and/or expression in a host cell.             -   The composition of the transgene sequence will depend                 upon the use to which the resulting vector will be put.                 For example, one type of transgene sequence includes a                 reporter sequence, which upon expression produces a                 detectable signal. Such reporter sequences include,                 without limitation, DNA sequences encoding β-lactamase,                 β-galactosidase (LacZ), alkaline phosphatase, thymidine                 kinase, green fluorescent protein (GFP), chloramphenicol                 acetyltransferase (CAT), luciferase, membrane bound                 proteins including, for example, CD2, CD4, CD8, the                 influenza hemagglutinin protein, and others well known                 in the art, to which high affinity antibodies directed                 thereto exist or can be produced by conventional means,                 and fusion proteins comprising a membrane bound protein                 appropriately fused to an antigen tag domain from, among                 others, hemagglutinin or Myc.             -   These coding sequences, when associated with regulatory                 elements which drive their expression, provide signals                 detectable by conventional means, including enzymatic,                 radiographic, colorimetric, fluorescence or other                 spectrographic assays, fluorescent activating cell                 sorting assays and immunological assays, including                 enzyme linked immunosorbent assay (ELISA),                 radioimmunoassay (RIA) and immunohistochemistry. For                 example, where the marker sequence is the LacZ gene, the                 presence of the vector carrying the signal is detected                 by assays for beta-galactosidase activity. Where the                 transgene is green fluorescent protein or luciferase,                 the vector carrying the signal may be measured visually                 by color or light production in a luminometer.             -   However, desirably, the transgene is a non-marker                 sequence encoding a product which is useful in biology                 and medicine, such as proteins, peptides, RNA, enzymes,                 dominant negative mutants, or catalytic RNAs. Desirable                 RNA molecules include tRNA, dsRNA, ribosomal RNA,                 catalytic RNAs, siRNA, small hairpin RNA, trans-splicing                 RNA, and antisense RNAs. One example of a useful RNA                 sequence is a sequence which inhibits or extinguishes                 expression of a targeted nucleic acid sequence in the                 treated animal. Typically, suitable target sequences in                 include oncologic targets and viral diseases. See, for                 examples of such targets the oncologic targets and                 viruses identified below in the section relating to                 immunogens.             -   The transgene may be used to correct or ameliorate gene                 deficiencies, which may include deficiencies in which                 normal genes are expressed at less than normal levels or                 deficiencies in which the functional gene product is not                 expressed. A preferred type of transgene sequence                 encodes a therapeutic protein or polypeptide which is                 expressed in a host cell. The invention further includes                 using multiple transgenes, e.g., to correct or                 ameliorate a gene defect caused by a multi-subunit                 protein. In certain situations, a different transgene                 may be used to encode each subunit of a protein, or to                 encode different peptides or proteins. This is desirable                 when the size of the DNA encoding the protein subunit is                 large, e.g., for an immunoglobulin, the platelet-derived                 growth factor, or a dystrophin protein. In order for the                 cell to produce the multi-subunit protein, a cell is                 infected with the recombinant virus containing each of                 the different subunits. Alternatively, different                 subunits of a protein may be encoded by the same                 transgene. In this case, a single transgene includes the                 DNA encoding each of the subunits, with the DNA for each                 subunit separated by an internal ribozyme entry site                 (IRES). This is desirable when the size of the DNA                 encoding each of the subunits is small, e.g., the total                 size of the DNA encoding the subunits and the IRES is                 less than five kilobases. As an alternative to an IRES,                 the DNA may be separated by sequences encoding a 2A                 peptide, which self-cleaves in a post-translational                 event. See, e.g., M. L. Donnelly, et al, J. Gen. Virol.,                 78(Pt 1):13-21 (Jan 1997); Furler, S., et al, Gene                 Ther., 8(11):864-873 (June 2001); Klump H., et al., Gene                 Ther., 8(10):811-817 (May 2001). This 2A peptide is                 significantly smaller than an IRES, making it well                 suited for use when space is a limiting factor. More                 often, when the transgene is large, consists of                 multi-subunits, or two transgenes are co-delivered, rAAV                 carrying the desired transgene(s) or subunits are                 co-administered to allow them to concatamerize in vivo                 to form a single vector genome. In such an embodiment, a                 first AAV may carry an expression cassette which                 expresses a single transgene and a second AAV may carry                 an expression cassette which expresses a different                 transgene for co-expression in the host cell. However,                 the selected transgene may encode any biologically                 active product or other product, e.g., a product                 desirable for study.             -   Suitable transgenes may be readily selected by one of                 skill in the art. The selection of the transgene is not                 considered to be a limitation of this invention.         -   2. Regulatory Elements             -   In addition to the major elements identified above for                 the minigene, the vector also includes conventional                 control elements which are operably linked to the                 transgene in a manner which permits its transcription,                 translation and/or expression in a cell transfected with                 the plasmid vector or infected with the virus produced                 by the invention. As used herein, “operably linked”                 sequences include both expression control sequences that                 are contiguous with the gene of interest and expression                 control sequences that act in trans or at a distance to                 control the gene of interest.             -   Expression control sequences include appropriate                 transcription initiation, termination, promoter and                 enhancer sequences; efficient RNA processing signals                 such as splicing and polyadenylation (polyA) signals;                 sequences that stabilize cytoplasmic mRNA; sequences                 that enhance translation efficiency (i.e., Kozak                 consensus sequence); sequences that enhance protein                 stability; and when desired, sequences that enhance                 secretion of the encoded product. A great number of                 expression control sequences, including promoters which                 are native, constitutive, inducible and/or                 tissue-specific, are known in the art and may be                 utilized.             -   Examples of constitutive promoters include, without                 limitation, the retroviral Rous sarcoma virus (RSV) LTR                 promoter (optionally with the RSV enhancer), the                 cytomegalovirus (CMV) promoter (optionally with the CMV                 enhancer) [see, e.g., Boshart et al, Cell, 41:521-530                 (1985)], the SV40 promoter, the dihydrofolate reductase                 promoter, the β-actin promoter, the phosphoglycerol                 kinase (PGK) promoter, and the EF1 promoter                 [Invitrogen]. Inducible promoters allow regulation of                 gene expression and can be regulated by exogenously                 supplied compounds, environmental factors such as                 temperature, or the presence of a specific physiological                 state, e.g., acute phase, a particular differentiation                 state of the cell, or in replicating cells only.                 Inducible promoters and inducible systems are available                 from a variety of commercial sources, including, without                 limitation, Invitrogen, Clontech and Ariad. Many other                 systems have been described and can be readily selected                 by one of skill in the art. Examples of inducible                 promoters regulated by exogenously supplied compounds,                 include, the zinc-inducible sheep metallothionine (MT)                 promoter, the dexamethasone (Dex)-inducible mouse                 mammary tumor virus (MMTV) promoter, the T7 polymerase                 promoter system [WO 98/10088]; the ecdysone insect                 promoter [No et al, Proc. Natl. Acad. Sci. USA,                 93:3346-3351 (1996)], the tetracycline-repressible                 system [Gossen et al, Proc. Natl. Acad. Sci. USA,                 89:5547-5551 (1992)], the tetracycline-inducible system                 [Gossen et al, Science, 268:1766-1769 (1995), see also                 Harvey et al, Curr. Opin. Chem. Biol., 2:512-518                 (1998)], the RU486-inducible system [Wang et al, Nat.                 Biotech., 15:239-243 (1997) and Wang et al, Gene Ther.,                 4:432-441 (1997)] and the rapamycin-inducible system                 [Magari et al, J. Clin. Invest., 100:2865-2872 (1997)].                 Other types of inducible promoters which may be useful                 in this context are those which are regulated by a                 specific physiological state, e.g., temperature, acute                 phase, a particular differentiation state of the cell,                 or in replicating cells only.             -   In another embodiment, the native promoter for the                 transgene will be used. The native promoter may be                 preferred when it is desired that expression of the                 transgene should mimic the native expression. The native                 promoter may be used when expression of the transgene                 must be regulated temporally or developmentally, or in a                 tissue-specific manner, or in response to specific                 transcriptional stimuli. In a further embodiment, other                 native expression control elements, such as enhancer                 elements, polyadenylation sites or Kozak consensus                 sequences may also be used to mimic the native                 expression.             -   Another embodiment of the transgene includes a gene                 operably linked to a tissue-specific promoter. For                 instance, if expression in skeletal muscle is desired, a                 promoter active in muscle should be used. These include                 the promoters from genes encoding skeletal β-actin,                 myosin light chain 2A, dystrophin, muscle creatine                 kinase, as well as synthetic muscle promoters with                 activities higher than naturally-occurring promoters                 (see Li et al., Nat. Biotech., 17:241-245 (1999)).                 Examples of promoters that are tissue-specific are known                 for liver (albumin, Miyatake et al., J Virol.,                 71:5124-32 (1997); hepatitis B virus core promoter,                 Sandig et al., Gene Ther., 3:1002-9 (1996);                 alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene                 Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et                 al., Mol. Biol. Rep., 24:185-96 (1997)); bone                 sialoprotein (Chen et al., J. Bone Miner. Res.,                 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J.                 Immunol., 161:1063-8 (1998); immunoglobulin heavy chain;                 T cell receptor chain), neuronal such as neuron-specific                 enolase (NSE) promoter (Andersen et al., Cell. Mol.                 Neurobiol., 13:503-15 (1993)), neurofilament light-chain                 gene (Piccioli et al., Proc. Natl. Acad. Sci. USA,                 88:5611-5 (1991)), and the neuron-specific vgf gene                 (Piccioli et al., Neuron, 15:373-84 (1995)), among                 others.             -   Optionally, plasmids carrying therapeutically useful                 transgenes may also include selectable markers or                 reporter genes may include sequences encoding geneticin,                 hygromicin or purimycin resistance, among others. Such                 selectable reporters or marker genes (preferably located                 outside the viral genome to be rescued by the method of                 the invention) can be used to signal the presence of the                 plasmids in bacterial cells, such as ampicillin                 resistance. Other components of the plasmid may include                 an origin of replication. Selection of these and other                 promoters and vector elements are conventional and many                 such sequences are available [see, e.g., Sambrook et al,                 and references cited therein].             -   The combination of the transgene, promoter/enhancer, and                 5′ and 3′ ITRs is referred to as a “minigene” for ease                 of reference herein. Provided with the teachings of this                 invention, the design of such a minigene can be made by                 resort to conventional techniques.         -   3. Delivery of the Minigene to a Packaging Host Cell             -   The minigene can be carried on any suitable vector,                 e.g., a plasmid, which is delivered to a host cell. The                 plasmids useful in this invention may be engineered such                 that they are suitable for replication and, optionally,                 integration in prokaryotic cells, mammalian cells, or                 both. These plasmids (or other vectors carrying the 5′                 AAV ITR-heterologous molecule-3′ITR) contain sequences                 permitting replication of the minigene in eukaryotes                 and/or prokaryotes and selection markers for these                 systems. Selectable markers or reporter genes may                 include sequences encoding geneticin, hygromicin or                 purimycin resistance, among others. The plasmids may                 also contain certain selectable reporters or marker                 genes that can be used to signal the presence of the                 vector in bacterial cells, such as ampicillin                 resistance. Other components of the plasmid may include                 an origin of replication and an amplicon, such as the                 amplicon system employing the Epstein Barr virus nuclear                 antigen. This amplicon system, or other similar amplicon                 components permit high copy episomal replication in the                 cells. Preferably, the molecule carrying the minigene is                 transfected into the cell, where it may exist                 transiently. Alternatively, the minigene (carrying the                 5′ AAV ITR-heterologous molecule-3′ ITR) may be stably                 integrated into the genome of the host cell, either                 chromosomally or as an episome. In certain embodiments,                 the minigene may be present in multiple copies,                 optionally in head-to-head, head-to-tail, or                 tail-to-tail concatamers. Suitable transfection                 techniques are known and may readily be utilized to                 deliver the minigene to the host cell.         -   Generally, when delivering the vector comprising the             minigene by transfection, the vector is delivered in an             amount from about 5 μg to about 100 μg DNA, about 10 to             about 50 μg DNA to about 1×10⁴ cells to about 1×10¹³ cells,             or about 10⁵ cells. However, the relative amounts of vector             DNA to host cells may be adjusted, taking into consideration             such factors as the selected vector, the delivery method and             the host cells selected.     -   B. Rep and Cap Sequences         -   In addition to the minigene, the host cell contains the             sequences which drive expression of the AAV8 capsid protein             (or a capsid protein comprising a fragment of the AAV8             capsid) in the host cell and rep sequences of the same             serotype as the serotype of the AAV ITRs found in the             minigene, or a cross-complementing serotype. The AAV cap and             rep sequences may be independently obtained from an AAV             source as described above and may be introduced into the             host cell in any manner known to one in the art as described             above. Additionally, when pseudotyping an AAV vector in an             AAV8 capsid, the sequences encoding each of the essential             rep proteins may be supplied by AAV8, or the sequences             encoding the rep proteins may be supplied by different AAV             serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,             AAV9). For example, the rep78/68 sequences may be from AAV2,             whereas the rep52/40 sequences may be from AAV8.         -   In one embodiment, the host cell stably contains the capsid             protein under the control of a suitable promoter, such as             those described above. Most desirably, in this embodiment,             the capsid protein is expressed under the control of an             inducible promoter. In another embodiment, the capsid             protein is supplied to the host cell in trans. When             delivered to the host cell in trans, the capsid protein may             be delivered via a plasmid which contains the sequences             necessary to direct expression of the selected capsid             protein in the host cell. Most desirably, when delivered to             the host cell in trans, the plasmid carrying the capsid             protein also carries other sequences required for packaging             the rAAV, e.g., the rep sequences.         -   In another embodiment, the host cell stably contains the rep             sequences under the control of a suitable promoter, such as             those described above. Most desirably, in this embodiment,             the essential rep proteins are expressed under the control             of an inducible promoter. In another embodiment, the rep             proteins are supplied to the host cell in trans. When             delivered to the host cell in trans, the rep proteins may be             delivered via a plasmid which contains the sequences             necessary to direct expression of the selected rep proteins             in the host cell. Most desirably, when delivered to the host             cell in trans, the plasmid carrying the capsid protein also             carries other sequences required for packaging the rAAV,             e.g., the rep and cap sequences.         -   Thus, in one embodiment, the rep and cap sequences may be             transfected into the host cell on a single nucleic acid             molecule and exist stably in the cell as an episome. In             another embodiment, the rep and cap sequences are stably             integrated into the chromosome of the cell. Another             embodiment has the rep and cap sequences transiently             expressed in the host cell. For example, a useful nucleic             acid molecule for such transfection comprises, from 5′ to             3′, a promoter, an optional spacer interposed between the             promoter and the start site of the rep gene sequence, an AAV             rep gene sequence, and an AAV cap gene sequence.         -   Optionally, the rep and/or cap sequences may be supplied on             a vector that contains other DNA sequences that are to be             introduced into the host cells. For instance, the vector may             contain the rAAV construct comprising the minigene. The             vector may comprise one or more of the genes encoding the             helper functions, e.g., the adenoviral proteins E1, E2a, and             E4ORF6, and the gene for VAI RNA.         -   Preferably, the promoter used in this construct may be any             of the constitutive, inducible or native promoters known to             one of skill in the art or as discussed above. In one             embodiment, an AAV P5 promoter sequence is employed. The             selection of the AAV to provide any of these sequences does             not limit the invention.         -   In another preferred embodiment, the promoter for rep is an             inducible promoter, such as are discussed above in             connection with the transgene regulatory elements. One             preferred promoter for rep expression is the T7 promoter.             The vector comprising the rep gene regulated by the T7             promoter and the cap gene, is transfected or transformed             into a cell which either constitutively or inducibly             expresses the T7 polymerase. See WO 98/10088, published Mar.             12, 1998.         -   The spacer is an optional element in the design of the             vector. The spacer is a DNA sequence interposed between the             promoter and the rep gene ATG start site. The spacer may             have any desired design; that is, it may be a random             sequence of nucleotides, or alternatively, it may encode a             gene product, such as a marker gene. The spacer may contain             genes which typically incorporate start/stop and polyA             sites. The spacer may be a non-coding DNA sequence from a             prokaryote or eukaryote, a repetitive non-coding sequence, a             coding sequence without transcriptional controls or a coding             sequence with transcriptional controls. Two exemplary             sources of spacer sequences are the phage ladder sequences             or yeast ladder sequences, which are available commercially,             e.g., from Gibco or Invitrogen, among others. The spacer may             be of any size sufficient to reduce expression of the rep78             and rep68 gene products, leaving the rep52, rep40 and cap             gene products expressed at normal levels. The length of the             spacer may therefore range from about 10 bp to about 10.0             kbp, preferably in the range of about 100 bp to about 8.0             kbp. To reduce the possibility of recombination, the spacer             is preferably less than 2 kbp in length; however, the             invention is not so limited.         -   Although the molecule(s) providing rep and cap may exist in             the host cell transiently (i.e., through transfection), it             is preferred that one or both of the rep and cap proteins             and the promoter(s) controlling their expression be stably             expressed in the host cell, e.g., as an episome or by             integration into the chromosome of the host cell. The             methods employed for constructing embodiments of this             invention are conventional genetic engineering or             recombinant engineering techniques such as those described             in the references above. While this specification provides             illustrative examples of specific constructs, using the             information provided herein, one of skill in the art may             select and design other suitable constructs, using a choice             of spacers, P5 promoters, and other elements, including at             least one translational start and stop signal, and the             optional addition of polyadenylation sites.         -   In another embodiment of this invention, the rep or cap             protein may be provided stably by a host cell.     -   C. The Helper Functions         -   The packaging host cell also requires helper functions in             order to package the rAAV of the invention. Optionally,             these functions may be supplied by a herpesvirus. Most             desirably, the necessary helper functions are each provided             from a human or non-human primate adenovirus source, such as             those described above and/or are available from a variety of             sources, including the American Type Culture Collection             (ATCC), Manassas, Va. (US). In one currently preferred             embodiment, the host cell is provided with and/or contains             an E1a gene product, an E1b gene product, an E2a gene             product, and/or an E4 ORF6 gene product. The host cell may             contain other adenoviral genes such as VAI RNA, but these             genes are not required. In a preferred embodiment, no other             adenovirus genes or gene functions are present in the host             cell.         -   By “adenoviral DNA which expresses the E1a gene product”, it             is meant any adenovirus sequence encoding E1a or any             functional E1a portion. Adenoviral DNA which expresses the             E2a gene product and adenoviral DNA which expresses the E4             ORF6 gene products are defined similarly. Also included are             any alleles or other modifications of the adenoviral gene or             functional portion thereof Such modifications may be             deliberately introduced by resort to conventional genetic             engineering or mutagenic techniques to enhance the             adenoviral function in some manner, as well as naturally             occurring allelic variants thereof. Such modifications and             methods for manipulating DNA to achieve these adenovirus             gene functions are known to those of skill in the art.         -   The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products,             as well as any other desired helper functions, can be             provided using any means that allows their expression in a             cell. Each of the sequences encoding these products may be             on a separate vector, or one or more genes may be on the             same vector. The vector may be any vector known in the art             or disclosed above, including plasmids, cosmids and viruses.             Introduction into the host cell of the vector may be             achieved by any means known in the art or as disclosed             above, including transfection, infection, electroporation,             liposome delivery, membrane fusion techniques, high velocity             DNA-coated pellets, viral infection and protoplast fusion,             among others. One or more of the adenoviral genes may be             stably integrated into the genome of the host cell, stably             expressed as episomes, or expressed transiently. The gene             products may all be expressed transiently, on an episome or             stably integrated, or some of the gene products may be             expressed stably while others are expressed transiently.             Furthermore, the promoters for each of the adenoviral genes             may be selected independently from a constitutive promoter,             an inducible promoter or a native adenoviral promoter. The             promoters may be regulated by a specific physiological state             of the organism or cell (i.e., by the differentiation state             or in replicating or quiescent cells) or by exogenously             added factors, for example.     -   D. Host Cells And Packaging Cell Lines         -   The host cell itself may be selected from any biological             organism, including prokaryotic (e.g., bacterial) cells, and             eukaryotic cells, including, insect cells, yeast cells and             mammalian cells. Particularly desirable host cells are             selected from among any mammalian species, including,             without limitation, cells such as A549, WEHI, 3T3, 10T1/2,             BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38,             HeLa, 293 cells (which express functional adenoviral E1),             Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast,             hepatocyte and myoblast cells derived from mammals including             human, monkey, mouse, rat, rabbit, and hamster. The             selection of the mammalian species providing the cells is             not a limitation of this invention; nor is the type of             mammalian cell, i.e., fibroblast, hepatocyte, tumor cell,             etc. The requirements for the cell used is that it not carry             any adenovirus gene other than E1, E2a and/or E4 ORF6; it             not contain any other virus gene which could result in             homologous recombination of a contaminating virus during the             production of rAAV; and it is capable of infection or             transfection of DNA and expression of the transfected DNA.             In a preferred embodiment, the host cell is one that has rep             and cap stably transfected in the cell.         -   One host cell useful in the present invention is a host cell             stably transformed with the sequences encoding rep and cap,             and which is transfected with the adenovirus E1, E2a, and             E4ORF6 DNA and a construct carrying the minigene as             described above. Stable rep and/or cap expressing cell             lines, such as B-50 (PCT/US98/19463), or those described in             U.S. Pat. No. 5,658,785, may also be similarly employed.             Another desirable host cell contains the minimum adenoviral             DNA which is sufficient to express E4 ORF6. Yet other cell             lines can be constructed using the AAV8 rep and/or AAV8 cap             sequences of the invention.         -   The preparation of a host cell according to this invention             involves techniques such as assembly of selected DNA             sequences. This assembly may be accomplished utilizing             conventional techniques. Such techniques include cDNA and             genomic cloning, which are well known and are described in             Sambrook et al., cited above, use of overlapping             oligonucleotide sequences of the adenovirus and AAV genomes,             combined with polymerase chain reaction, synthetic methods,             and any other suitable methods which provide the desired             nucleotide sequence.         -   Introduction of the molecules (as plasmids or viruses) into             the host cell may also be accomplished using techniques             known to the skilled artisan and as discussed throughout the             specification. In preferred embodiment, standard             transfection techniques are used, e.g., CaPO₄ transfection             or electroporation, and/or infection by hybrid             adenovirus/AAV vectors into cell lines such as the human             embryonic kidney cell line HEK 293 (a human kidney cell line             containing functional adenovirus E1 genes which provides             trans-acting E1 proteins).         -   The AAV8 based vectors which are generated by one of skill             in the art are beneficial for gene delivery to selected host             cells and gene therapy patients since no neutralization             antibodies to AAV8 have been found in the human population.             One of skill in the art may readily prepare other rAAV viral             vectors containing the AAV8 capsid proteins provided herein             using a variety of techniques known to those of skill in the             art. One may similarly prepare still other rAAV viral             vectors containing AAV8 sequence and AAV capsids of another             serotype.         -   One of skill in the art will readily understand that the             AAV8 sequences of the invention can be readily adapted for             use in these and other viral vector systems for in vitro, ex             vivo or in vivo gene delivery. Similarly, one of skill in             the art can readily select other fragments of the AAV8             genome of the invention for use in a variety of rAAV and             non-rAAV vector systems. Such vectors systems may include,             e.g., lentiviruses, retroviruses, poxviruses, vaccinia             viruses, and adenoviral systems, among others. Selection of             these vector systems is not a limitation of the present             invention.         -   Thus, the invention further provides vectors generated using             the nucleic acid and amino acid sequences of the novel AAV             of the invention. Such vectors are useful for a variety of             purposes, including for delivery of therapeutic molecules             and for use in vaccine regimens. Particularly desirable for             delivery of therapeutic molecules are recombinant AAV             containing capsids of the novel AAV of the invention. These,             or other vector constructs containing novel AAV sequences of             the invention may be used in vaccine regimens, e.g., for             co-delivery of a cytokine, or for delivery of the immunogen             itself.

V. Recombinant Viruses And Uses Therefor

Using the techniques described herein, one of skill in the art can generate a rAAV having a capsid of a serotype 8 of the invention or having a capsid containing one or more fragments of AAV8. In one embodiment, a full-length capsid from a single serotype, e.g., AAV8 [SEQ ID NO: 2] can be utilized. In another embodiment, a full-length capsid may be generated which contains one or more fragments of AAV8 fused in frame with sequences from another selected AAV serotype, or from heterologous portions of AAV8. For example, a rAAV may contain one or more of the novel hypervariable region sequences of AAV8. Alternatively, the unique AAV8 sequences of the invention may be used in constructs containing other viral or non-viral sequences. Optionally, a recombinant virus may carry AAV8 rep sequences encoding one or more of the AAV8 rep proteins.

A. Delivery of Viruses

-   -   In another aspect, the present invention provides a method for         delivery of a transgene to a host which involves transfecting or         infecting a selected host cell with a recombinant viral vector         generated with the AAV8 sequences (or functional fragments         thereof) of the invention. Methods for delivery are well known         to those of skill in the art and are not a limitation of the         present invention.

In one desirable embodiment, the invention provides a method for AAV8 mediated delivery of a transgene to a host. This method involves transfecting or infecting a selected host cell with a recombinant viral vector containing a selected transgene under the control of sequences which direct expression thereof and AAV8 capsid proteins.

Optionally, a sample from the host may be first assayed for the presence of antibodies to a selected AAV serotype. A variety of assay formats for detecting neutralizing antibodies are well known to those of skill in the art. The selection of such an assay is not a limitation of the present invention. See, e.g., Fisher et al, Nature Med., 3(3):306-312 (March 1997) and W C Manning et al, Human Gene Therapy, 9:477-485 (Mar. 1, 1998). The results of this assay may be used to determine which AAV vector containing capsid proteins of a particular serotype are preferred for delivery, e.g., by the absence of neutralizing antibodies specific for that capsid serotype.

In one aspect of this method, the delivery of vector with AAV8 capsid proteins may precede or follow delivery of a gene via a vector with a different serotype AAV capsid protein. Thus, gene delivery via rAAV vectors may be used for repeat gene delivery to a selected host cell. Desirably, subsequently administered rAAV vectors carry the same transgene as the first rAAV vector, but the subsequently administered vectors contain capsid proteins of serotypes which differ from the first vector. For example, if a first vector has AAV8 capsid proteins, subsequently administered vectors may have capsid proteins selected from among the other serotypes.

Optionally, multiple rAAV8 vectors can be used to deliver large transgenes or multiple transgenes by co-administration of rAAV vectors concatamerize in vivo to form a single vector genome. In such an embodiment, a first AAV may carry an expression cassette which expresses a single transgene (or a subunit thereof) and a second AAV may carry an expression cassette which expresses a second transgene (or a different subunit) for co-expression in the host cell. A first AAV may carry an expression cassette which is a first piece of a polycistronic construct (e.g., a promoter and transgene, or subunit) and a second AAV may carry an expression cassette which is a second piece of a polycistronic construct (e.g., transgene or subunit and a polyA sequence). These two pieces of a polycistronic construct concatamerize in vivo to form a single vector genome which co-expresses the transgenes delivered by the first and second AAV. In such embodiments, the rAAV vector carrying the first expression cassette and the rAAV vector carrying the second expression cassette can be delivered in a single pharmaceutical composition. In other embodiments, the two or more rAAV vectors are delivered as separate pharmaceutical compositions which can be administered substantially simultaneously, or shortly before or after one another.

The above-described recombinant vectors may be delivered to host cells according to published methods. The rAAV, preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a desired organ (e.g., the liver (optionally via the hepatic artery) or lung), oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired.

Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a therapeutically effective human dosage of the viral vector is generally in the range of from about 0.1 ml to about 100 ml of solution containing concentrations of from about 1×10⁹ to 1×10¹⁶ genomes virus vector. A preferred human dosage for delivery to large organs (e.g., liver, muscle, heart and lung) may be about 5×10¹⁰ to 5×10¹³ AAV genomes per 1 kg, at a volume of about 1 to 100 mL. A preferred dosage for delivery to eye is about 5×10⁹ to 5×10¹² genome copies, at a volume of about 0.1 mL to 1 mL. The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The levels of expression of the transgene can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the minigene. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention.

Examples of therapeutic products and immunogenic products for delivery by the AAV8-containing vectors of the invention are provided below. These vectors may be used for a variety of therapeutic or vaccinal regimens, as described herein. Additionally, these vectors may be delivered in combination with one or more other vectors or active ingredients in a desired therapeutic and/or vaccinal regimen.

-   B. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor a superfamily, including TGFα, activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, any one of the heregluin/neuregulin/ARIA/neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, any one of the family of semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system are also useful in the invention. These include, without limitations, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, as well as engineered immunoglobulins and MHC molecules. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins. The invention encompasses receptors for cholesterol regulation and/or lipid modulation, including the low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, the very low density lipoprotein (VLDL) receptor, and scavenger receptors. The invention also encompasses gene products such as members of the steroid hormone receptor superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D receptors and other nuclear receptors. In addition, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilms tumor protein, ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence. Still other useful gene products include enzymes such as may be useful in enzyme replacement therapy, which is useful in a variety of conditions resulting from deficient activity of enzyme. For example, enzymes that contain mannose-6-phosphate may be utilized in therapies for lysosomal storage diseases (e.g., a suitable gene includes that encoding β-glucuronidase (GUSB)).

Still other useful gene products include those used for treatment of hemophilia, including hemophilia B (including Factor IX) and hemophilia A (including Factor VIII and its variants, such as the light chain and heavy chain of the heterodimer and the B-deleted domain; U.S. Pat. No. 6,200,560 and U.S. Pat. No. 6,221,349). The Factor VIII gene codes for 2351 amino acids and the protein has six domains, designated from the amino to the terminal carboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al, Nature, 312:330 (1984); Vehar et al., Nature 312:337 (1984); and Toole et al, Nature, 342:337 (1984)]. Human Factor VIII is processed within the cell to yield a heterodimer primarily comprising a heavy chain containing the A1, A2 and B domains and a light chain containing the A3, C1 and C2 domains. Both the single chain polypeptide and the heterodimer circulate in the plasma as inactive precursors, until activated by thrombin cleavage between the A2 and B domains, which releases the B domain and results in a heavy chain consisting of the A1 and A2 domains. The B domain is deleted in the activated procoagulant form of the protein. Additionally, in the native protein, two polypeptide chains (“a” and “b”), flanking the B domain, are bound to a divalent calcium cation.

In some embodiments, the minigene comprises first 57 base pairs of the Factor VIII heavy chain which encodes the 10 amino acid signal sequence, as well as the human growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the minigene further comprises the A1 and A2 domains, as well as 5 amino acids from the N-terminus of the B domain, and/or 85 amino acids of the C-terminus of the B domain, as well as the A3, C1 and C2 domains. In yet other embodiments, the nucleic acids encoding Factor VIII heavy chain and light chain are provided in a single minigene separated by 42 nucleic acids coding for 14 amino acids of the B domain [U.S. Pat. No. 6,200,560].

As used herein, a therapeutically effective amount is an amount of AAV vector that produces sufficient amounts of Factor VIII to decrease the time it takes for a subject's blood to clot. Generally, severe hemophiliacs having less than 1% of normal levels of Factor VIII have a whole blood clotting time of greater than 60 minutes as compared to approximately 10 minutes for non-hemophiliacs.

The present invention is not limited to any specific Factor VIII sequence. Many natural and recombinant forms of Factor VIII have been isolated and generated. Examples of naturally occurring and recombinant forms of Factor VII can be found in the patent and scientific literature including, U.S. Pat. No. 5,563,045, U.S. Pat. No. 5,451,521, U.S. Pat. No. 5,422,260, U.S. Pat. No. 5,004,803, U.S. Pat. No. 4,757,006, U.S. Pat. No. 5,661,008, U.S. Pat. No. 5,789,203, U.S. Pat. No. 5,681,746, U.S. Pat. No. 5,595,886, U.S. Pat. No. 5,045,455, U.S. Pat. No. 5,668,108, U.S. Pat. No. 5,633,150, U.S. Pat. No. 5,693,499, U.S. Pat. No. 5,587,310, U.S. Pat. No. 5,171,844, U.S. Pat. No. 5,149,637, U.S. Pat. No. 5,112,950, U.S. Pat. No. 4,886,876, WO 94/11503, WO 87/07144, WO 92/16557, WO 91/09122, WO 97/03195, WO 96/21035, WO 91/07490, EP 0 672 138, EP 0 270 618, EP 0 182 448, EP 0 162 067, EP 0 786 474, EP 0 533 862, EP 0 506 757, EP 0 874 057, EP 0 795 021, EP 0 670 332, EP 0 500 734, EP 0 232 112, EP 0 160 457, Sanberg et al., XXth Int. Congress of the World Fed. Of Hemophilia (1992), and Lind et al., Eur. J. Biochem., 232:19 (1995).

Nucleic acids sequences coding for the above-described Factor VIII can be obtained using recombinant methods or by deriving the sequence from a vector known to include the same. Furthermore, the desired sequence can be isolated directly from cells and tissues containing the same, using standard techniques, such as phenol extraction and PCR of cDNA or genomic DNA [See, e.g., Sambrook et al]. Nucleotide sequences can also be produced synthetically, rather than cloned. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence [See, e.g., Edge, Nature 292:757 (1981); Nambari et al, Science, 223:1299 (1984); and Jay et al, J. Biol. Chem. 259:6311 (1984).

Furthermore, the invention is not limited to human Factor VIII. Indeed, it is intended that the present invention encompass Factor VIII from animals other than humans, including but not limited to companion animals (e.g., canine, felines, and equines), livestock (e.g., bovines, caprines and ovines), laboratory animals, marine mammals, large cats, etc.

The AAV vectors may contain a nucleic acid coding for fragments of Factor VIII which is itself not biologically active, yet when administered into the subject improves or restores the blood clotting time. For example, as discussed above, the Factor VIII protein comprises two polypeptide chains: a heavy chain and a light chain separated by a B-domain which is cleaved during processing. As demonstrated by the present invention, co-tranducing recipient cells with the Factor VIII heavy and light chains leads to the expression of biologically active Factor VIII. Because, however, most hemophiliacs contain a mutation or deletion in only one of the chain (e.g., heavy or light chain), it may be possible to administer only the chain defective in the patient to supply the other chain.

Other useful gene products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides having a non-naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions. For example, single-chain engineered immunoglobulins could be useful in certain immunocompromised patients. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which could be used to reduce overexpression of a target.

Reduction and/or modulation of expression of a gene is particularly desirable for treatment of hyperproliferative conditions characterized by hyperproliferating cells, as are cancers and psoriasis. Target polypeptides include those polypeptides which are produced exclusively or at higher levels in hyperproliferative cells as compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk and EGRF. In addition to oncogene products as target antigens, target polypeptides for anti-cancer treatments and protective regimens include variable regions of antibodies made by B cell lymphomas and variable regions of T cell receptors of T cell lymphomas which, in some embodiments, are also used as target antigens for autoimmune disease. Other tumor-associated polypeptides can be used as target polypeptides such as polypeptides which are found at higher levels in tumor cells including the polypeptide recognized by monoclonal antibody 17-1A and folate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those which may be useful for treating individuals suffering from autoimmune diseases and disorders by conferring a broad based protective immune response against targets that are associated with autoimmunity including cell receptors and cells which produce “self”-directed antibodies. T cell mediated autoimmune diseases include Rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease and ulcerative colitis. Each of these diseases is characterized by T cell receptors (TCRs) that bind to endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases.

-   C. Immunogenic Transgenes

Suitably, the AAV8 vectors of the invention avoid the generation of immune responses to the AAV8 sequences contained within the vector. However, these vectors may nonetheless be formulated in a manner which permits the expression of a transgene carried by the vectors to induce an immune response to a selected antigen. For example, in order to promote an immune response, the transgene may be expressed from a constitutive promoter, the vector can be adjuvanted as described herein, and/or the vector can be put into degenerating tissue.

Examples of suitable immunogenic transgenes include those selected from a variety of viral families. Example of desirable viral families against which an immune response would be desirable include, the picornavirus family, which includes the genera rhinoviruses, which are responsible for about 50% of cases of the common cold; the genera enteroviruses, which include polioviruses, coxsackieviruses, echoviruses, and human enteroviruses such as hepatitis A virus; and the genera apthoviruses, which are responsible for foot and mouth diseases, primarily in non-human animals. Within the picornavirus family of viruses, target antigens include the VP1, VP2, VP3, VP4, and VPG. Other viral families include the astroviruses and the calcivirus family. The calcivirus family encompasses the Norwalk group of viruses, which are an important causative agent of epidemic gastroenteritis. Still another viral family desirable for use in targeting antigens for inducing immune responses in humans and non-human animals is the togavirus family, which includes the genera alphavirus, which include Sindbis viruses, RossRiver virus, and Venezuelan, Eastern & Western Equine encephalitis, and rubivirus, including Rubella virus. The flaviviridae family includes dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis and tick borne encephalitis viruses. Other target antigens may be generated from the Hepatitis C or the coronavirus family, which includes a number of non-human viruses such as infectious bronchitis virus (poultry), porcine transmissible gastroenteric virus (pig), porcine hemagglutinatin encephalomyelitis virus (pig), feline infectious peritonitis virus (cats), feline enteric coronavirus (cat), canine coronavirus (dog), and human respiratory coronaviruses, which may cause the common cold and/or non-A, B or C hepatitis, and which include the putative cause of sudden acute respiratory syndrome (SARS). Within the coronavirus family, target antigens include the E1 (also called M or matrix protein), E2 (also called S or Spike protein), E3 (also called HE or hemagglutin-elterose) glycoprotein (not present in all coronaviruses), or N (nucleocapsid). Still other antigens may be targeted against the arterivirus family and the rhabdovirus family. The rhabdovirus family includes the genera vesiculovirus (e.g., Vesicular Stomatitis Virus), and the general lyssavirus (e.g., rabies). Within the rhabdovirus family, suitable antigens may be derived from the G protein or the N protein. The family filoviridae, which includes hemorrhagic fever viruses such as Marburg and Ebola virus may be a suitable source of antigens. The paramyxovirus family includes parainfluenza Virus Type 1, parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3, rubulavirus (mumps virus, parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastle disease virus (chickens), rinderpest, morbillivirus, which includes measles and canine distemper, and pneumovirus, which includes respiratory syncytial virus. The influenza virus is classified within the family orthomyxovirus and is a suitable source of antigen (e.g., the HA protein, the N1 protein). The bunyavirus family includes the genera bunyavirus (California encephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus (puremala is a hemahagin fever virus), nairovirus (Nairobi sheep disease) and various unassigned bungaviruses. The arenavirus family provides a source of antigens against LCM and Lassa fever virus. Another source of antigens is the bornavirus family. The reovirus family includes the genera reovirus, rotavirus (which causes acute gastroenteritis in children), orbiviruses, and cultivirus (Colorado Tick fever, Lebombo (humans), equine encephalosis, blue tongue). The retrovirus family includes the sub-family oncorivirinal which encompasses such human and veterinary diseases as feline leukemia virus, HTLVI and HTLVII, lentivirinal (which includes HIV, simian immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, and spumavirinal). The papovavirus family includes the sub-family polyomaviruses (BKU and JCU viruses) and the sub-family papillomavirus (associated with cancers or malignant progression of papilloma). The adenovirus family includes viruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/or enteritis. The parvovirus family feline parvovirus (feline enteritis), feline panleucopeniavirus, canine parvovirus, and porcine parvovirus. The herpesvirus family includes the sub-family alphaherpesvirinae, which encompasses the genera simplexvirus (HSVI, HSVII), Varicellovirus (pseudorabies, Varicella zoster) and the sub-family betaherpesvirinae, which includes the genera cytomegalovirus (HCMV, muromegalovirus) and the sub-family gammaherpesvirinae, which includes the genera lymphocryptovirus, EBV (Burkitts lymphoma), human herpesviruses 6A, 6B and 7, Kaposi's sarcoma-associated herpesvirus and cercopithecine herpesvirus (B virus), infectious rhinotracheitis, Marek's disease virus, and rhadinovirus. The poxvirus family includes the sub-family chordopoxvirinae, which encompasses the genera Orthopoxvirus (Variola major (Smallpox) and Vaccinia (Cowpox)), parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and the sub-family entomopoxvirinae. The hepadnavirus family includes the Hepatitis B virus. One unclassified virus which may be suitable source of antigens is the Hepatitis delta virus, Hepatitis E virus, and prions. Another virus which is a source of antigens is Nipan Virus. Still other viral sources may include avian infectious bursal disease virus and porcine respiratory and reproductive syndrome virus. The alphavirus family includes equine arteritis virus and various Encephalitis viruses.

The present invention may also encompass immunogens which are useful to immunize a human or non-human animal against other pathogens including bacteria, fungi, parasitic microorganisms or multicellular parasites which infect human and non-human vertebrates, or from a cancer cell or tumor cell. Examples of bacterial pathogens include pathogenic gram-positive cocci include pneumococci; staphylococci (and the toxins produced thereby, e.g., enterotoxin B); and streptococci. Pathogenic gram-negative cocci include meningococcus; gonococcus. Pathogenic enteric gram-negative bacilli include enterobacteriaceae; Pseudomonas, acinetobacteria and eikenella; melioidosis; salmonella; shigella; haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella species (brucellosis); Francisella tularensis (which causes tularemia); Yersinia pestis (plague) and other Yersinia (Pasteurella); Streptobacillus moniliformis and spirillum; Gram-positive bacilli include Listeria monocytogenes; Erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria); cholera; B. anthracis (anthrax); donovanosis (granuloma inguinale); and bartonellosis. Diseases caused by pathogenic anaerobic bacteria include tetanus; botulism (Clostridum botulinum and its toxin); Clostridium perfringens and its epsilon toxin; other clostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochetal diseases include syphilis; treponematoses: yaws, pinta and endemic syphilis; and leptospirosis. Other infections caused by higher pathogen bacteria and pathogenic fungi include glanders (Burkholderia mallei); actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma and chromomycosis; and dermatophytosis. Rickettsial infections include Typhus fever, Rocky Mountain spotted fever, Q fever (Coxiella burnetti), and Rickettsialpox. Examples of mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections. Pathogenic eukaryotes encompass pathogenic protozoans and helminths and infections produced thereby include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm) infections.

Many of these organisms and/or the toxins produced thereby have been identified by the Centers for Disease Control [(CDC), Department of Heath and Human Services, USA], as agents which have potential for use in biological attacks. For example, some of these biological agents, include, Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague), variola major (smallpox), Francisella tularensis (tularemia), and viral hemorrhagic fevers [filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa, Machupo]), all of which are currently classified as Category A agents; Coxiella burnetti (Q fever); brucella species (brucellosis), Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis), Ricinus communis and its toxin (ricin toxin), Clostridium perfringens and its toxin (epsilon toxin), Staphylococcus species and their toxins (enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats (e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsia powazekii), and viral encephalitis (alphaviruses, e.g., Venezuelan equine encephalitis; eastern equine encephalitis; western equine encephalitis); all of which are currently classified as Category B agents; and Nipan virus and hantaviruses, which are currently classified as Category C agents. In addition, other organisms, which are so classified or differently classified, may be identified and/or used for such a purpose in the future. It will be readily understood that the viral vectors and other constructs described herein are useful to deliver antigens from these organisms, viruses, their toxins or other by-products, which will prevent and/or treat infection or other adverse reactions with these biological agents.

Administration of the vectors of the invention to deliver immunogens against the variable region of the T cells elicit an immune response including CTLs to eliminate those T cells. In rheumatoid arthritis (RA), several specific variable regions of TCRs which are involved in the disease have been characterized. These TCRs include V-3, V-14, V-17 and V-17. Thus, delivery of a nucleic acid sequence that encodes at least one of these polypeptides will elicit an immune response that will target T cells involved in RA. In multiple sclerosis (MS), several specific variable regions of TCRs which are involved in the disease have been characterized. These TCRs include V-7 and V-10. Thus, delivery of a nucleic acid sequence that encodes at least one of these polypeptides will elicit an immune response that will target T cells involved in MS. In scleroderma, several specific variable regions of TCRs which are involved in the disease have been characterized. These TCRs include V-6, V-8, V-14 and V-16, V-3C, V-7, V-14, V-15, V-16, V-28 and V-12. Thus, delivery of a nucleic acid molecule that encodes at least one of these polypeptides will elicit an immune response that will target T cells involved in scleroderma.

Thus, a rAAV8-derived recombinant viral vector of the invention provides an efficient gene transfer vehicle which can deliver a selected transgene to a selected host cell in vivo or ex vivo even where the organism has neutralizing antibodies to one or more AAV serotypes. In one embodiment, the rAAV and the cells are mixed ex vivo; the infected cells are cultured using conventional methodologies; and the transduced cells are re-infused into the patient.

These compositions are particularly well suited to gene delivery for therapeutic purposes and for immunization, including inducing protective immunity. Further, the compositions of the invention may also be used for production of a desired gene product in vitro. For in vitro production, a desired product (e.g., a protein) may be obtained from a desired culture following transfection of host cells with a rAAV containing the molecule encoding the desired product and culturing the cell culture under conditions which permit expression. The expressed product may then be purified and isolated, as desired. Suitable techniques for transfection, cell culturing, purification, and isolation are known to those of skill in the art.

The following examples illustrate several aspects and embodiments of the invention.

Example 1: Production of Recombinant AAV8 Viral Genomes Equipped With AAV2 ITRs

Chimeric packaging constructs are generated by fusing AAV2 rep with cap sequences of novel AAV serotypes. These chimeric packaging constructs are used, initially, for pseudotyping recombinant AAV genomes carrying AAV2 ITRs by triple transfection in 293 cell using Ad5 helper plasmid. These pseudotyped vectors are used to evaluate performance in transduction-based serological studies and evaluate gene transfer efficiency of novel AAV serotypes in different animal models including NHP and rodents, before intact and infectious viruses of these novel serotypes are isolated.

A. pAAV2GFP

-   -   The AAV2 plasmid which contains the AAV2 ITRs and green         fluorescent protein expressed under the control of a         constitutive promoter. This plasmid contains the following         elements: the AAV2 ITRs, a CMV promoter and the GFP coding         sequences.

B. Cloning of Trans Plasmid

-   -   To construct the chimeric trans-plasmid for production of         recombinant pseudotyped AAV8 vectors, p5E18 plasmid (Xiao et         al., 1999, J Virol 73:3994-4003) was partially digested with Xho         Ito linearize the plasmid at the Xho I site at the position of         3169 bp only. The Xho I cut ends were then filled in and ligated         back. This modified p5E18 plasmid was restricted with Xba I and         Xho I in a complete digestion to remove the AAV2 cap gene         sequence and replaced with a 2267 bp Spe I/Xho I fragment         containing the AAV8 cap gene which was isolated from pCRAAV8         6-5+15-4 plasmid.     -   The resulting plasmid contains the AAV2 rep sequences for         Rep78/68 under the control of the AAV2 P5 promoter, and the AAV2         rep sequences for Rep52/40 under the control of the AAV2 P19         promoter. The AAV9 capsid sequences are under the control of the         AAV2 P40 promoter, which is located within the Rep sequences.         This plasmid further contains a spacer 5′ of the rep ORF.     -   Alternatively, a similar plasmid can be constructed which         utilizes the AAV8 rep sequences and the native AAV8 promoter         sequences. This plasmid is then used for production of rAAV8, as         described herein.

C. Production of Pseudotyped rAAV

-   -   The rAAV particles (AAV2 vector in AAV8 capsid) are generated         using an adenovirus-free method. Briefly, the cis plasmid         (pAAV2.1 lacZ plasmid containing AAV2 ITRs), and the trans         plasmid pCRAAV8 6-5+15-4 (containing the AAV2 rep and AAV8cap)         and a helper plasmid, respectively, were simultaneously         co-transfected into 293 cells in a ratio of 1:1:2 by calcium         phosphate precipitation.     -   For the construction of the pAd helper plasmids, pBG10 plasmid         was purchased from Microbix (Canada). A RsrII fragment         containing L2 and L3 was deleted from pBHG10, resulting in the         first helper plasmid, pAdΔ F13. Plasmid Ad F1 was constructed by         cloning Asp700/SalI fragment with a PmeI/Sgfl deletion,         isolating from pBHG10, into Bluescript. MLP, L2, L2 and L3 were         deleted in the pAdΔF1. Further deletions of a 2.3 kb Nrul         fragment and, subsequently, a 0.5 kb RsrII/NruI fragment         generated helper plasmids pAdΔF5 and pAdΔF6, respectively. The         helper plasmid, termed pΔF6, provides the essential helper         functions of E2a and E4 ORF6 not provided by the E1-expressing         helper cell, but is deleted of adenoviral capsid proteins and         functional E1 regions).     -   Typically, 50 μg of DNA (cis:trans:helper) was transfected onto         a 150 mm tissue culture dish. The 293 cells were harvested 72         hours post-transfection, sonicated and treated with 0.5% sodium         deoxycholate (37° C. for 10 min.) Cell lysates were then         subjected to two rounds of a CsCl gradient. Peak fractions         containing rAAV vector are collected, pooled and dialyzed         against PBS.

Example 2—Evaluation of Vectors with AAV8 Capsids

Vectors based on AAV1 (2/1), AAVS (2/5) and AAV2 (2/2) were developed essentially as described for AAV8 in Example 1. Genome copy (GC) titers of AAV vectors were determined by TaqMan analysis using probes and primers targeting SV40 poly A region as described previously [Gao, G., et al., (2000) Hum Gene Ther 11, 2079-91]. Recombinant virions were recovered by CsCl₂ sedimentation in all cases except AAV2/2, which was purified by heparin chromatography.

Vectors were constructed for each serotype for a number of in vitro and in vivo studies. Eight different transgene cassettes were incorporated into the vectors and recombinant virions were produced for each serotype. The recovery of virus, based on genome copies, is summarized in Table 1. The yields of vector were high for each serotype with no consistent differences between serotypes. Data presented in the table are average genome copy yields with standard deviation×10¹³ of multiple production lots of 50 plate (150 mm) transfections.

TABLE 1 Production of Recombinant Vectors AAV2/1 AAV2/2 AAV2/5 AAV2/8 CMV 7.30 ± 4.33 4.49 ± 2.89 5.19 ± 5.19 0.87 LacZ (n = 9) (n = 6) (n = 8) (n = 1) CMV 6.43 ± 2.42 3.39 ± 2.42 5.55 ± 6.49 3.74 ± 3.88 EGFP (n = 2) (n = 2) (n = 4) (n = 2) TBG 4.18  0.23 0.704 ± 0.43   0.532 LacZ (n = 1) (n = 1) (n = 2) (n = 1) Alb 4.67 ± 0.75 4.77 4.09 2.02 A1AT (n = 2) (n = 1) (n = 1) (n = 1) CB 0.567  0.438 2.82 0.816 ± 0.679 A1AT (n = 1) (n = 1) (n = 1) (n = 2) CMV 8.78 ± 2.37 1.43 ± 1.18 1.63 ± 1.15 1.32 ± 0.87 rhCG (n = 7) (n = 2) (n = 3) (n = 3) TBG 8.51 ± 6.65 3.47 ± 2.09 5.26 ± 3.85 1.83 ± 0.98 rhCG (n = 6) (n = 5) (n = 4) (n = 5) TBG 1.24 ± 1.29  0.63 ± 0.394 3.74 ± 2.48 15.8 ± 15.0 cFIX (n = 3) (n = 6) (n = 7) (n = 5)

Example 3—Serologic Analysis of Pseudotyped Vectors

C57BL/6 mice were injected with vectors of different serotypes of AAVCBA1AT vectors intramuscularly (5×10¹¹ GC) and serum samples were collected 34 days later. To test neutralizing and cross-neutralizing activity of sera to each serotype of AAV, sera was analyzed in a transduction based neutralizing antibody assay [Gao, G. P., et al., (1996) J Virol 70, 8934-43]. More specifically, the presence of neutralizing antibodies was determined by assessing the ability of serum to inhibit transduction of 84-31 cells by reporter viruses (AAVCMVEGFP) of different serotypes. Specifically, the reporter virus AAVCMVEGFP of each serotype [at multiplicity of infection (MOI) that led to a transduction of 90% of indicator cells] was pre-incubated with heat-inactivated serum from animals that received different serotypes of AAV or from naïve mice. After 1-hour incubation at 37° C., viruses were added to 84-31 cells in 96 well plates for 48 or 72-hour, depending on the virus serotype. Expression of GFP was measured by FluoroImagin (Molecular Dynamics) and quantified by Image Quant Software. Neutralizing antibody titers were reported as the highest serum dilution that inhibited transduction to less than 50%.

The availability of GFP expressing vectors simplified the development of an assay for neutralizing antibodies that was based on inhibition of transduction in a permissive cell line (i.e., 293 cells stably expressing E4 from Ad5). Sera to selected AAV serotypes were generated by intramuscular injection of the recombinant viruses. Neutralization of AAV transduction by 1:20 and 1:80 dilutions of the antisera was evaluated (Table 2). Antisera to AAV1, AAV2, AAVS and AAV8 neutralized transduction of the serotype to which the antiserum was generated (AAVS and AAV8 to a lesser extent than AAV1 and AAV2) but not to the other serotype (i.e., there was no evidence of cross neutralization suggesting that AAV 8 is a truly unique serotype).

TABLE 2 Serological Analysis of New AAV Serotypes. Immunization Serum dilution: Serum dilution: Serum dilution: Serum dilution: Sera: Vector 1/20 1/80 1/20 1/80 1/20 1/80 1/20 1/80 Group 1 AAV2/1 0 0 100 100 100 100 100 100 Group 2 AAV2/2 100 100 0 0 100 100 100 100 Group 3 AAV2/5 100 100 100 100 16.5 16.5 100 100 Group 4 AAV2/8 100 100 100 100 100 100 26.3 60

Human sera from 52 normal subjects were screened for neutralization against selected serotypes. No serum sample was found to neutralize AAV2/8 while AAV2/2 and AAV2/1 vectors were neutralized in 20% and 10% of sera, respectively. A fraction of human pooled IqG representing a collection of 60,000 individual samples did not neutralize AAV2/8, whereas AAV2/2 and AAV2/1 vectors were neutralized at titers of serum equal to 1/1280 and 1/640, respectively.

Example 4—In Vivo Evaluation of Different Serotypes of AAV Vectors

In this study, 7 recombinant AAV genomes, AAV2CBhA1AT, AAV2AlbhA1AT, AAV2CMVrhCG, AAV2TBGrhCG, AAV2TBGcFIX, AAV2CMVLacZ and AAV2TBGLacZ were packaged with capsid proteins of different serotypes. In all 7 constructs, minigene cassettes were flanked with AAV2 ITRs. cDNAs of human α-antitrypsin (A1AT) [Xiao, W., et al., (1999) J Virol 73, 3994-4003] β-subunit of rhesus monkey choriogonadotropic hormone (CG) [Zoltick, P. W. & Wilson, J. M. (2000) Mol Ther 2, 657-9] canine factor IX [Wang, L., et al., (1997) Proc Natl Acad Sci USA 94, 11563-6] and bacterial β-glactosidase (i.e., Lac Z) genes were used as reporter genes. For liver-directed gene transfer, either mouse albumin gene promoter (Alb) [Xiao, W. (1999), cited above] or human thyroid hormone binding globulin gene promoter (TBG) [Wang (1997), cited above] was used to drive liver specific expression of reporter genes. In muscle-directed gene transfer experiments, either cytomegalovirus early promoter (CMV) or chicken β-actin promoter with CMV enhancer (CB) was employed to direct expression of reporters.

For muscle-directed gene transfer, vectors were injected into the right tibialis anterior of 4-6 week old NCR nude or C57BL/6 mice (Taconic, Germantown, N.Y.) at a dose of 1×10¹¹ genome copies (GC) per animal In liver-directed gene transfer studies, vectors were infused intraportally into 7-9 week old NCR nude or C57BL/6 mice (Taconic, Germantown, N.Y.), also at a dose of 1×10¹¹ genome copies (GC) per animal Serum samples were collected intraorbitally at different time points after vector administration. Muscle and liver tissues were harvested at different time points for cryosectioning and Xgal histochemical staining from animals that received the lacZ vectors. For the re-administration experiment, C56BL/6 mice initially received AAV2/1, 2/2, 2/5, 2/7 and 2/8CBA1AT vectors intramuscularly and followed for A1AT gene expression for 7 weeks. Animals were then treated with AAV2/8TBGcFIX intraportally and studied for cFIX gene expression.

ELISA based assays were performed to quantify serum levels of hA1AT, rhCG and cFIX proteins as described previously [Gao, G. P., et al., (1996) J Virol 70, 8934-43; Zoltick, P. W. & Wilson, J. M. (2000) Mol Ther 2, 657-9; Wang, L., et al., Proc Natl Acad Sci USA 94, 11563-6]. The experiments were completed when animals were sacrificed for harvest of muscle and liver tissues for DNA extraction and quantitative analysis of genome copies of vectors present in target tissues by TaqMan using the same set of primers and probe as in titration of vector preparations [Zhang, Y., et al., (2001) Mol Ther 3, 697-707].

The performance of vectors base on the new serotypes were evaluated in murine models of muscle and liver-directed gene transfer and compared to vectors based on the known serotypes AAV1, AAV2 and AAV5. Vectors expressing secreted proteins (A1AT and CG-Table 3) were used to quantitate relative transduction efficiencies between different serotypes through ELISA analysis of sera. The cellular distribution of transduction within the target organ was evaluated using lacZ expressing vectors and X-gal histochemistry .

The performance of AAV vectors in skeletal muscle was analyzed following direct injection into the tibialis anterior muscles. Vectors contained the same AAV2 based genome with the immediate early gene of CMV or a CMV enhanced β-actin promoter driving expression of the transgene. Previous studies indicated that immune competent C57BL/6 mice elicit limited humoral responses to the human A1AT protein when expressed from AAV vectors [Xiao, W., et al., (1999) J Virol 73, 3994-4003].

In each strain, AAV2/1 vector produced the highest levels of A1AT and AAV2/2 vector the lowest, with AAV2/8 vectors showing intermediate levels of expression. Peak levels of CG at 28 days following injection of nu/nu NCR mice showed the highest levels from AAV2/7 and the lowest from AAV2/2 with AAV2/8 and AAV2/1 in between. Injection of AAV2/1 lacZ vectors yielded gene expression at the injection sites in all muscle fibers with substantially fewer lacZ positive fibers observed with AAV2/2 and AAV 2/8 vectors.

Similar murine models were used to evaluate liver-directed gene transfer. Identical doses of vector based on genome copies were infused into the portal veins of mice that were analyzed subsequently for expression of the transgene. Each vector contained an AAV2 based genome using previously described liver-specific promoters (i.e., albumin or thyroid hormone binding globulin) to drive expression of the transgene. More particularly, CMVCG and TBGCG minigene cassettes were used for muscle and liver-directed gene transfer, respectively. Levels of rhCG were defined as relative units (rUs×10³). The data were from assaying serum samples collected at day 28, post vector administration (4 animals per group). As shown in Table 4, the impact of capsid proteins on the efficiency of transduction of A1AT vectors in nu/nu and C57BL/6 mice and CG vectors in C57BL/6 mice was consistent, i.e., AAV2/8 is the most efficient for pseudotype for liver-directed gene transfer.

TABLE 3 Expression of β-unit of Rhesus Monkey Chorionic Gonadotropin (rhCG) in Mouse Muscle and Liver. Vector Muscle Liver AAV2/1 4.5 ± 2.1 1.6 ± 1.0 AAV2 0.5 ± 0.1 0.7 ± 0.3 AAV2/5 ND* 4.8 ± 0.8 AAV2/8 4.0 ± 0.7 76.0 ± 22.8 *Not determined in this experiment.

In all cases, AAV2/8 vectors yielded the highest levels of transgene expression that ranged from 16 to 110 greater than what was obtained with AAV2/2 vectors; expression from AAV2/5 was intermediate. Analysis of X-Gal stained liver sections of animals that received the corresponding lacZ vectors showed a correlation between the number of transduced cells and overall levels of transgene expression. DNAs extracted from livers of C57BL/6 mice who received the A1AT vectors were analyzed for abundance of vector DNA using real time PCR technology.

The amount of vector DNA found in liver 56 days after injection correlated with the levels of transgene expression (Table 4). For this experiment, a set of probe and primers targeting the SV40 polyA region of the vector genome was used for TaqMan PCR. Values shown are means of three individual animals with standard deviations. The animals were sacrificed at day 56 to harvest liver tissues for DNA extraction. These studies indicate that AAV8 is the most efficient vector for liver-directed gene transfer due to increased numbers of transduced hepatocytes.

TABLE 4 Real Time PCR Analysis for Abundance of AAV Vectors in nu/nu Mouse Liver Following Injection of 1 × 10¹¹ Genome Copies of Vector. AAV vectors/Dose Genome Copies per Cell AAV2/1AlbA1AT  0.6 ± 0.36 AAV2AlbA1AT 0.003 ± 0.001 AAV2/5AlbA1AT 0.83 ± 0.64 AAV2/8AlbA1AT 18 ± 11

The serologic data described above suggest that AAV2/8 vector should not be neutralized in vivo following immunization with the other serotypes. C57BL/6 mice received intraportal injections of AAV2/8 vector expressing canine factor IX (10¹¹ genome copies) 56 days after they received intramuscular injections of A1AT vectors of different serotypes. High levels of factor IX expression were obtained 14 days following infusion of AAV2/8 into naïve animals (17+2 μg/ml, N=4) which were not significantly different that what was observed in animals immunized with AAV2/1 (31+23 μg/ml, N=4), and AAV2/2 (16 μg/ml, N=2). This contrasts to what was observed in AAV2/8 immunized animals that were infused with the AAV2/8 factor IX vector in which no detectable factor IX was observed (<0.1 μg/ml, N=4).

Oligonucleotides to conserved regions of the cap gene did amplify sequences from rhesus monkeys that represented unique AAVs. Identical cap signature sequences were found in multiple tissues from rhesus monkeys derived from at least two different colonies. Full-length rep and cap open reading frames were isolated and sequenced from single sources. Only the cap open reading frames of the novel AAVs were necessary to evaluate their potential as vectors because vectors with the AAV8 capsids were generated using the ITRs and rep from AAV2. This also simplified the comparison of different vectors since the actual vector genome is identical between different vector serotypes. In fact, the yields of recombinant vectors generated using this approach did not differ between serotypes.

Vectors based on AAV8 appear to be immunologically distinct (i.e., they are not neutralized by antibodies generated against other serotypes). Furthermore, sera from humans do not neutralize transduction by AAV8 vectors, which is a substantial advantage over the human derived AAVs currently under development for which a significant proportion of the human population has pre-existing immunity that is neutralizing [Chirmule, N., et al., (1999) Gene Ther 6, 1574-83].

The tropism of the new vector is favorable for in vivo applications. Importantly, AAV2/8 provides a substantial advantage over the other serotypes in terms of efficiency of gene transfer to liver that until now has been relatively disappointing in terms of the numbers of hepatocytes stably transduced. AAV2/8 consistently achieved a 10 to 100-fold improvement in gene transfer efficiency as compared to the other vectors. The basis for the improved efficiency of AAV2/8 is unclear, although it presumably is due to uptake via a different receptor that is more active on the basolateral surface of hepatocytes. This improved efficiency will be quite useful in the development of liver-directed gene transfer where the number of transduced cells is critical, such as in urea cycle disorders and familial hypercholesterolemia.

Thus, the lack of pre-existing immunity to AAV8 and the favorable tropism of the vectors for liver indicates that vectors with AAV8 capsid proteins are suitable for use as vectors in human gene therapy and other in vivo applications.

Example 5—Tissue Tropism Studies

In the design of a high throughput functional screening scheme for novel AAV constructs, a non-tissue specific and highly active promoter, CB promoter (CMV enhanced chicken β-actin promoter) was selected to drive an easily detectable and quantifiable reporter gene, human α-anti-trypsin gene. Thus only one vector for each new AAV clone needs to be made for gene transfer studies targeting 3 different tissues, liver, lung and muscle to screen for tissue tropism of a particular AAV construct. The following table summarizes data generated from novel AAV vectors in the tissue tropism studies (AAVCBA1AT). Table 5 reports data obtained (in μg A1AT/mL serum) at day 14 of the study.

TABLE 5 Target Tissue Vector Lung Liver Muscle AAV2/1 ND ND 45 ± 11 AAV2/5 0.6 ± 0.2 ND ND AAV2/8 ND 84 ± 30 ND AAV vector carried CC10hA1AT minigene for lung specific expression were pseudotyped with capsids of novel AAVs were given to Immune deficient animals (NCR nude) in equal volume (50 μl each of the original preps without dilution) via intratracheal injections as provided in the following table. The vectors were also administered to immune competent animals (C57BL/6) in equal genome copies (1×10¹¹ GC) as shown in the Table 6. (1×10¹¹ GC per animal, C57BL/6, day 14, detection limit≧0.033 μg/ml). As shown, AAV8 is the best liver transducer.

TABLE 6 μg of A1AT/ml AAV Vector with 1 × 10¹¹ vector 2/1 0.076 ± 0.031 2/2  0.1 ± 0.09 2/5 0.0840.033 2/8 1.92 ± 1.3 

Example 6 —Model of Hypercholesterolemia

To further assess the effect of rAAV-mediated transgene expression by the AAV2/8 constructs of the invention, a further study was performed.

A. Vector Construction

-   -   AAV vectors packaged with AAV8 capsid proteins were constructed         using a pseudotyping strategy [Hildinger M, et al., J Virol         2001; 75:6199-6203]. Recombinant AAV genomes with AAV2 inverted         terminal repeats (ITR) were packaged by triple transfection of         293 cells with the cis-plasmid, the adenovirus helper plasmid         and a chimeric packaging construct, a fusion of the capsids of         the novel AAV serotypes with the rep gene of AAV2. The chimeric         packaging plasmid was constructed as previously described         [Hildinger et al, cited above]. The recombinant vectors were         purified by the standard CsCl₂ sedimentation method. To         determine the yield TaqMan (Applied Biosystems) analysis was         performed using probes and primers targeting the SV40 poly(A)         region of the vectors [Gao GP, et al., Hum Gene Ther. 2000 Oct         10;11(15):2079-91]. The resulting vectors express the transgene         under the control of the human thyroid hormone binding globulin         gene promoter (TBG).

B. Animals

-   -   LDL receptor deficient mice on the C57B1/6 background were         purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and         maintained as a breeding colony. Mice were given unrestricted         access to water and obtained a high fat Western Diet (high %         cholesterol) starting three weeks prior vector injection. At day         −7 as well at day 0, blood was obtained via retroorbital bleeds         and the lipid profile evaluated. The mice were randomly divided         into seven groups. The vector was injected via an intraportal         injection as previously described ([Chen S J et al., Mol Therapy         2000; 2(3), 256-261]. Briefly, the mice were anaesthetized with         ketamine and xylazine. A laparotomy was performed and the portal         vein exposed. Using a 30 g needle the appropriate dose of vector         diluted in 100 μl PBS was directly injected into the portal         vein. Pressure was applied to the injection site to ensure a         stop of the bleeding. The skin wound was closed and draped and         the mice carefully monitored for the following day. Weekly         bleeds were performed starting at day 14 after liver directed         gene transfer to measure blood lipids. Two animals of each group         were sacrificed at the timepoints week 6 and week 12 after         vector injection to examine atherosclerotic plaque size as well         as receptor expression. The remaining mice were sacrificed at         week 20 for plaque measurement and determination of transgene         expression.

TABLE 7 Vector dose N Group 1 AAV2/8-TBG-hLDLr 1 × 10¹² gc 12 Group 2 AAV2/8-TBG-hLDLr 3 × 10¹¹ gc 12 Group 3 AAV2/8-TBG-hLDLr 1 × 10¹¹ gc 12

C. Serum Lipoprotein and Liver Function Analysis

-   -   Blood samples were obtained from the retroorbital plexus after a         6 hour fasting period. The serum was separated from the plasma         by centrifugation. The amount of plasma lipoproteins and liver         transaminases in the serum were detected using an automatized         clinical chemistry analyzer (ACE, Schiapparelli Biosystems,         Alpha Wassermann)

D. Detection of Transgene Expression

-   -   LDL receptor expression was evaluated by immuno-fluorescence         staining and Western blotting. For Western Blot frozen liver         tissue was homogenized with lysis buffer (20 mM Tris, pH 7.4,         130 mM NaCl, 1% Triton X 100, proteinase inhibitor complete,         EDTA-free, Roche, Mannheim, Germany). Protein concentration was         determined using the Micro BCA Protein Assay Reagent Kit         (Pierce, Rockford, Ill.). 40 μg of protein was resolved on 4-15%         Tris-HCl Ready Gels (Biorad, Hercules, Calif.) and transferred         to a nitrocellulose membrane (Invitrogen). To generate anti-hLDL         receptor antibodies a rabbit was injected intravenously with an         AdhLDLr prep (1×10¹³ gc). Four weeks later the rabbit serum was         obtained and used for Western Blot. A 1:100 dilution of the         serum was used as a primary antibody followed by a         HRP-conjugated anti-rabbit IgG and ECL chemiluminescent         detection (ECL Western Blot Detection Kit, Amersham, Arlington         Heights, Ill.).

D. Immunocytochemistry

-   -   For determination of LDL receptor expression in frozen liver         sections immunohistochemistry analyses were performed. 10 um         cryostat sections were either fixed in acetone for 5 minutes, or         unfixed. Blocking was obtained via a 1 hour incubation period         with 10% of goat serum. Sections were then incubated for one         hour with the primary antibody at room temperature. A rabbit         polyclonal antibody anti-human LDL (Biomedical Technologies         Inc., Stoughton, Mass.) was used diluted accordingly to the         instructions of the manufacturer. The sections were washed with         PBS, and incubated with 1:100 diluted fluorescein goat         anti-rabbit IgG (Sigma, St Louis, Mo.). Specimens were finally         examined under fluorescence microscope Nikon Microphot-FXA. In         all cases, each incubation was followed by extensive washing         with PBS. Negative controls consisted of preincubation with PBS,         omission of the primary antibody, and substitution of the         primary antibody by an isotype-matched non-immune control         antibody. The three types of controls mentioned above were         performed for each experiment on the same day.

E. Gene Transfer Efficiency

-   -   Liver tissue was obtained after sacrificing the mice at the         designated time points. The tissue was shock frozen in liquid         nitrogen and stored at −80° C. until further processing. DNA was         extracted from the liver tissue using a QIAamp DNA Mini Kit         (QIAGEN GmbH, Germany) according to the manufacturers protocol.         Genome copies of AAV vectors in the liver tissue were evaluated         using Taqman analysis using probes and primers against the SV40         poly(A) tail as described above.

F. Atherosclerotic Plaque Measurement

-   -   For the quantification of the atherosclerotic plaques in the         mouse aorta the mice were anaesthetized (10% ketamine and         xylazine, ip), the chest opened and the arterial system perfused         with ice-cold phosphate buffered saline through the left         ventricle. The aorta was then carefully harvested, slit down         along the ventral midline from the aortic arch down to the         femoral arteries and fixed in formalin. The lipid-rich         atherosclerotic plaques were stained with Sudan IV (Sigma,         Germany) and the aorta pinned out flat on a black wax surface.         The image was captured with a Sony DXC-960 MD color video         camera. The area of the plaque as well as of the complete aortic         surface was determined using Phase 3 Imaging Systems (Media         Cybernetics).

G. Clearance off I¹²⁵ LDL

-   -   Two animals per experimental group were tested. A bolus of         I¹²⁵-labeled LDL (generously provided by Dan Rader, Upenn) was         infused slowly through the tail vein over a period of 30 sec         (1,000,000 counts of [I¹²⁵ ]-LDL diluted in 100 μl sterile         PBS/animal) At time points 3 min, 30 min, 1.5 hr, 3 hr, 6 hr         after injection a blood sample was obtained via the         retro-orbital plexus. The plasma was separated off from the         whole blood and 10 μl plasma counted in the gamma counter.         Finally the fractional catabolic rate was calculated from the         lipoprotein clearance data.

H. Evaluation of Liver Lipid Accumulation

-   -   Oil Red Staining of frozen liver sections was performed to         determine lipid accumulation. The frozen liver sections were         briefly rinsed in distilled water followed by a 2 minute         incubation in absolute propylene glycol. The sections were then         stained in oil red solution (0.5% in propylene glycol) for 16         hours followed by counterstaining with Mayer's hematoxylin         solution for 30 seconds and mounting in warmed glycerin jelly         solution.     -   For quantification of the liver cholesterol and triglyceride         content liver sections were homogenized and incubated in         chloroform/methanol (2:1) overnight. After adding of 0.05% H₂SO₄         and centrifugation for 10 minutes, the lower layer of each         sample was collected, divided in two aliquots and dried under         nitrogen. For the cholesterol measurement the dried lipids of         the first aliquot were dissolved in 1% Triton X-100 in         chloroform. Once dissolved, the solution was dried under         nitrogen. After dissolving the lipids in ddH₂O and incubation         for 30 minutes at 37° C. the total cholesterol concentration was         measured using a Total Cholesterol Kit (Wako Diagnostics). For         the second aliquot the dried lipids were dissolved in alcoholic         KOH and incubated at 60° C. for 30 minutes. Then 1 M MgCl₂ was         added, followed by incubation on ice for 10 minutes and         centrifugation at 14,000 rpm for 30 minutes. The supernatant was         finally evaluated for triglycerides (Wako Diagnostics).     -   All of the vectors pseudotyped in an AAV2/8 capsid lowered total         cholesterol, LDL and triglycerides as compared to the control.         These test vectors also corrected phenotype of         hypercholesterolemia in a dose-dependent manner. A reduction in         plaque area for the AAV2/8 mice was observed in treated mice at         the first test (2 months), and the effect was observed to         persist over the length of the experiment (6 months).

Example 7—Functional Factor IX Expression and Correction of Hemophilia

A. Knock-Out Mice

-   -   Functional canine factor IX (FIX) expression was assessed in         hemophilia B mice. Vectors with capsids of AAV1, AAV2, AAVS or         AAV8 were constructed to deliver AAV2 5′ ITR—liver-specific         promoter [LSP]—canine FIX—woodchuck hepatitis post-regulatory         element (WPRE)—AAV2 3′ ITR . The vectors were constructed as         described in Wang et al, 2000, Molecular Therapy 2: 154-158),         using the appropriate capsids.     -   Knock-out mice were generated as described in Wang et al, 1997.         Proc. Natl. Acad. Sci. USA 94: 11563-11566. This model closely         mimic the phenotypes of hemophilia B in human.     -   Vectors of different serotypes were delivered as a single         intraportal injection into the liver of adult hemophiliac         C57B^(1/6) mice in a dose of 1×10¹¹ GC/mouse for the five         different serotypes and a second AAV8 vector was also delivered         at 1×10¹⁰ GC/mouse. Control group was injected with 1×10¹¹ GC of         AAV2/8 TBG LacZ3. Each group contains 5-10 male and female mice.         Mice were bled bi-weekly after vector administration.     -   1. ELISA         -   The canine FIX concentration in the mouse plasma was             determined by an ELISA assay specific for canine factor IX,             performed essentially as described by Axelrod et al, 1990,             Proc.Natl.Acad.Sci.USA, 87:5173-5177 with modifications.             Sheep anti-canine factor IX (Enzyme Research Laboratories)             was used as primary antibody and rabbit anti-canine factor             IX ((Enzyme Research Laboratories) was used as secondary             antibody. Beginning at two weeks following injection,             increased plasma levels of cFIX were detected for all test             vectors. The increased levels were sustained at therapeutic             levels throughout the length of the experiment, i.e., to 12             weeks. Therapeutic levels are considered to be 5% of normal             levels, i.e., at about 250 ng/mL.     -   The highest levels of expression were observed for the AAV2/8         (at 10¹¹), with sustained superphysiology levels cFIX levels         (ten-fold higher than the normal level). Expression levels for         AAV2/8 (10¹¹) were approximately 10 fold higher than those         observed for AAV2/2 and AAV2/8 (10¹⁰) . The lowest expression         levels, although still above the therapeutic range, were         observed for AAV2/5.     -   2. In Vitro Activated Partial Thromboplastin Time (aPTT) Assay         -   Functional factor IX activity in plasma of the FIX knock-out             mice was determined by an in vitro activated partial             thromboplastin time (aPTT) assay—Mouse blood samples were             collected from the retro-orbital plexus into 1/10 volume of             citrate buffer. APTT assay was performed as described by             Wang et al, 1997, Proc. Natl. Acad. Sci. USA 94:             11563-11566.         -   Clotting times by aPTT on plasma samples of all vector             injected mice were within the normal range (approximately 60             sec) when measured at two weeks post-injection, and             sustained clotting times in the normal or shorter than             normal range throughout the study period (12 weeks).         -   Lowest sustained clotting times were observed in the animals             receiving AAV2/8 (10¹¹). By week 12, AAV2/2 also induced             clotting times similar to those for AAV2/8. However, this             lowered clotting time was not observed for AAV2/2 until week             12, whereas lowered clotting times (in the 25-40 sec range)             were observed for AAV2/8 beginning at week two.         -   Immuno-histochemistry staining on the liver tissues             harvested from some of the treated mice is currently being             performed. About 70-80% of hepatocytes are stained positive             for canine FIX in the mouse injected with AAV2/8.cFIX             vector.

B. Hemophilia B Dogs

-   -   Dogs that have a point mutation in the catalytic domain of the         F.IX gene, which, based on modeling studies, appears to render         the protein unstable, suffer from hemophilia B [Evans et al,         1989, Proc. Natl. Acad. Sci. USA, 86:10095-10099). A colony of         such dogs has been maintained for more than two decades at the         University of North Carolina, Chapel Hill. The homeostatic         parameters of these doges are well described and include the         absence of plasma F.IX antigen, whole blood clotting times in         excess of 60 minutes, whereas normal dogs are 6-8 minutes, and         prolonged activated partial thromboplastin time of 50-80         seconds, whereas normal dogs are 13-28 seconds. These dogs         experience recurrent spontaneous hemorrhages. Typically,         significant bleeding episodes are successfully managed by the         single intravenous infusion of 10 ml/kg of normal canine plasma;         occasionally, repeat infusions are required to control bleeding.     -   Four dogs were injected intraportally with AAV.cFIX according to         the schedule below. A first dog received a single injection with         AAV2/2.cFIX at a dose of 3.7×10¹¹ genome copies (GC)/kg and was         sacrificed at day 665 due to severe spinal hemorrhage. A second         dog received a first injection of AAV2/2.cFIX (2.8×10¹¹ GC/kg),         followed by a second injection with AAV2/5.cFIX (2.3×10¹³ GC/kg)         at day 1180. A third dog received a single injection with         AAV2/2.cFIX at a dose of 4.6×10¹² GC/kg. The fourth dog received         an injection with AAV2/2.cFIX (2.8×10¹² GC/kg) and an injection         at day 995 with AAV2/8.cFIX (5×10¹² GC/kg).     -   The abdomen of hemophilia dogs were aseptically and surgically         opened under general anesthesia and a single infusion of vector         was administered into the portal vein. The animals were         protected from hemorrhage in the pen-operative period by         intravenous administration of normal canine plasma. The dog was         sedated, intubated to induce general anesthesia, and the abdomen         was shaved and prepped. After the abdomen was opened, the spleen         was moved into the operative field. The splenic vein was located         and a suture was loosely placed proximal to a small distal         incision in the vein. An introduced was rapidly inserted into         the vein, then the suture loosened and a 5 F cannula was         threaded to an intravenous location near the portal vein         threaded to an intravenous location near the portal vein         bifurcation. After hemostasis was secured and the catheter         balloon was inflated, approximately 5.0 ml of vector diluted in         PBS was infused into the portal vein over a 5 minute interval.         The vector infusion was followed by a 5.0 ml infusion of saline.         The balloon was then deflated, the callula was removed and         venous hemostatis was secured. The spleen was then replaced,         bleeding vessels were cauterized and the operative wound was         closed. The animal was extubated having tolerated the surgical         procedure well. Blood samples were analyzed as described. [Wang         et al, 2000, Molecular Therapy 2: 154-158]

The results are summarized in the table below. Dog C51, female, was 13.6 kg and 6.5 months old at the time of first injection. Dog C52, male, was 17.6 kg and 6.5 months old at first injection; and 17.2 kg and 45.2 months at second injection. Dog C55, male, was a 19.0 kg and 12.0 months at first injection. Dog D39, female, was a 5.0 kg and 2.8 months at first injection; 22.6 kg and 35.4 months old at the time of the second injection. In the table, GC refers to genome copies of the AAV vectors. WBCT were >60 minutes (except C52=42 min) before injection. Baseline aPTT for C51=98.4 sec, C52=97.7 sec; C55=145.1 sec; D39=97.8 sec. Bleeds post-treatment were spontaneous bleeding episodes happening in hemophilia B dogs post-AAV vector treatment that required treatment with plasma infusion.

TABLE 8 Hemophilia B Dogs Injected with rAAV intraportally Vector Avg Avg cFIX Dose Total GC WBCT Avg aPTT plasma Dog Vector (GC/kg) Inject (min) (min) (ng/mL) 1^(st) C51 AAV2- 3.7 × 10¹¹   5 × 10¹² 13.2 ± 2.1 77.5 ± 15.1 3.8 ± 1.0 injection LSP.cFIX C52 AAV2- 2.8 × 10¹¹ 5.0 × 10¹² 16.1 ± 3.5 81.5 ± 17.7 3.7 ± 1.1 LSP.cFIX C55 AAV2- 4.6 × 10¹² 8.7 × 10¹³ 10.2 ± 2.2 46.4 ± 6.1  259.7 ± 28.5  LSP.cFIX WPRE D39 AAV2- 2.8 × 10¹² 1.4 × 10¹³ 11.5 ± 2.6 59.1 ± 6.3  34.4 ± 9.8  LSPcFIX WPRE 2^(nd) C52 AAV2/5- 2.3 × 10¹³ 4.0 × 10¹⁴ 12.9 ± 1.1 41.9 ± 2.7  817.3 ± 102.1 injection LSP.cFIX WPRE D39 AAV2/8- 5.0 × 10¹² 1.1 × 10¹⁴ 12.6 ± 1.5 656.9 ± 1.1  LSP.cFIX WPRE

-   -   1. Whole Blood Clotting Time (WBCT)         -   WBCT following injection with the AAV2/2 vectors were             somewhat variable, ranging from about 6.5 min to 30 minutes.             WBCT for a normal dog is 6-12 min Sharp drops in WBCT were             observed immediately upon injection with the AAV2/8 or             AAV2/5 vectors The sharp drop was also observed in C55             injected with AAV2 (d2=9 min), and for C51 and C52, the             early data point for WBCT were not checked. The sharp drop             is believed to be due to the dog plasma infusion before and             after the surgery. WBCT is an assay very sensitive to low             level of FIX, it is not very sensitive to the actual level             of FIX (aPTT is more relevant).     -   2. aPTT Assay         -   Clotting times by aPTT on plasma samples of all vector             injected dogs were variable over the first approximately 700             days, at which time clotting times leveled in the normal             range (40-60 sec, normal dog: 24-32 sec). A sharp drop into             the normal range was observed following each of the second             injections (AAV2/8 or AAV2/5). While clotting times were not             sustained in the normal range, clotting times were reduced             to levels below those observed prior to the second             injection.         -   For aPTT, normal dogs are 24-32 sec, and hemophilia B dogs             are 80-106 sec. For C51 and C52 who received low dose of             AAV2.cFIX vector, average aPTT after treatment remain at             77.5 and 81.5 sec, not significantly different from             hemophilia B dogs without treatment. Higher dose of AAV2             improved the average aPTT to 59.1 and 46.4 sec, respectively             for D39 and C55. After the treatment of AAV2/5, the average             aPTT for C52 improved significantly from 81.5 sec to 41.9             sec. And for D39, after the AAV2/8 treatment, the average             aPTT improve from 59.1 sec.     -   3. Canine Factor IX ELISA         -   cFIX levels were detectable following the first set of             injections, albeit below therapeutic levels. Following             injection with AAV2/8 and AAV2/5, levels of cFIX rose spiked             into the therapeutic range and then leveled off within the             therapeutic range (normal is 5 μg/ml in plasma, therapeutic             level is 5% of normal level which is 250 ng/ml).         -   The first three weeks of WBCT, aPTT and cFIX antigen are             affected by the dog plasma infusion before and after the             surgery. It is hard to conclude the drop of clotting time or             the rise of cFIX antigen level is due to the vector or the             plasma infusion for the first 3 weeks. However, it is             interesting to note that the quick and dramatic rise of cFIX             antigen after 2/5 and 2/8 vector injection. This is unique             to AAV2/5 and 2/8 injected dogs and could be attributed to             AAV2/5 and 2/8 vectors rather than the normal dog plasma             infusion, since all dogs received similar amount of normal             dog plasma infusion for the surgery. Three days after AAV2/8             injection, the level of cFIX in the plasma of D39 reached             9.5 μg/ml and peaked at 10.4 μg/ml at day 6, twice as much             as the normal level (5 μg/ml). The cFIX level gradually             decreased to the average of 817ng/ml (C52, AAV2/5) and 657             ng/ml (D39, AAV2/8). In C52, 3 days after injection of             AAV2/5 vector, the cFIX level reached 2.6 μg/ml and peaked             at 4.6 μg/ml at day 7. In C55, who received AAV2 vector at             the dose similar to that of AAV2/8 injected to D39, peaked             only at 2.2 μg/ml at day 3, then gradually dropped and             maintained at 5% of normal level of cFIX.         -   The doses of vector received by C55 (AAV2, 4.6×10¹² GC/kg)             and the second injection in D39 (AAV2/8, 5×10¹² GC/kg) were             very close. However, the cFIX expression levels raised in             D39 by AAV2/8 vector (average 657-34=623 ng/ml, 12.5% of             normal level) was 2.5 fold higher than that in C55 (average             259 ng/ml, 5% of normal level). This suggests AAV2/8 is 2.5             fold more potent than AAV2 in dogs injected intraportally             with similar dose of vectors. And in the same dog D39, the             second injection of two fold higher dose of AAV2/8             dramatically increased the cFIX level from 0.7% to 13.1%,             18.7 fold higher than the first injection. And in C52, the             second injection of 2.3×10¹³ GC/ml of AAV2/5 vector resulted             in average 817 ng/ml (16.3% of normal level) of cFIX in the             plasma. This was only marginally higher (1.3 fold) than the             cFIX level raised in D39 by AAV2/8 (average 623 ng/ml, 12.5%             of normal level,). However, the dose of AAV2/5 injected in             C52 was 4.6 fold higher than the dose of AAV2/8 injected in             D39. This suggests that AAV2/8 vector is also more potent             than AAV2/5 vector in dogs.         -   The first injection of AAV2 vectors did not block the             success of transduction by AAV2/5 and AAV2/8 vectors after             the second injection in dogs. Readministration using a             different serotype of AAV vector can be used as an approach             to treat animals or humans who have been previously exposed             to AAV2 or treated with AAV2 vectors.

Example 8—Mouse Model of Liver Enzyme Disorder

The AAV2/8 vector generated as described herein was studied for its efficiency in transferring the liver enzyme gene ornithine transcarbamylase (OTC) in an accepted animal model for OTC deficiency [X. Ye et al, Pediatric Research, 41(4):527-534 (1997); X. Ye et al, J Biol. Chem., 271(7):3639-3646 (Feb. 1996)]. The results of this experiment (data not shown) demonstrate that an AAV2/8 vector of the invention carrying the ornithine transcarbamylase (OTC) gene was observed to correct OTC deficiency.

Example 9—In Vivo Expression of Factor VIII

Three groups of C57BL/6 mice are injected via the portal vein with either 3×10¹¹ genome copies AAV vector carrying the Factor VIII heavy chain (FVIII-HC), 3×10¹¹ genome copies of AAV vector carrying Factor VIII light chain (FVIII-LC), or 3×10¹¹ particles of both AAV-FVIII-HC and AAV-FVIII-LC. In addition, a group of four animals is injected with 3×10¹¹ particles of AAV carrying Factor IX (FIX), which is known to be useful in treatment of hemophilia B. It has been shown that this strain of mice does not elicit an immune response to human FVIII when the gene is delivered to the liver via an adenoviral vector (Connelly et al., Blood 87:4671-4677 [1996]).

These experiments will demonstrate the feasibility of producing biologically active FVIII using two AAV vectors to independently deliver the heavy and light chains of FVIII.

Blood samples are collected in sodium citrate via the retro-orbital plexus at biweekly intervals for the first 2 months and at monthly intervals thereafter for 6 months and at 11 months. Very high levels of FVIII light chain will be expressed in animals injected with AAV-FVIII-LC alone or both vectors.

In order to assess the amount of biologically active human FVIII produced in the animals, a modified ChromZ assay is used. Since this assay detects both human and murine FVIII, the amount of FVIII present in the plasma before and after adsorption to an antibody specific to human FVIII is determined. The amount of FVIII remaining in the plasma after adsorption represents the amount of active murine FVIII and the difference represented the amount of active human FVIII. The modified ChromZ assay will indicate that only those animals injected with both vectors produced biologically active FVIII.

The animals are expected to maintain physiological levels of active protein for more than 11 months, without waning.

All publications cited in this specification, and any sequence listings associated therewith, are incorporated herein by reference. While the invention has been described with reference to particularly preferred embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. 

1. An adeno-associated virus (AAV)8 viral vector comprising an AAV8 capsid having packaged therein a heterologous gene operably linked to regulatory sequences which direct its expression, wherein the heterologous gene encodes factor IX. 